Method of stabilizing molecules without refrigeration using water soluble polymers and applications thereof in performing chemical reactions

ABSTRACT

The present application is directed to methods of performing chemical reactions, including multi-step chemical reactions in which two or more of the reagents in the chemical reaction are incorporated or entrapped in a solid polymeric structure comprising pullulan. In certain embodiments, the chemical reaction or multi-step reaction serves as a sensor. Accordingly the present application is also directed to sensors for performing the methods of the application. In certain embodiments, at least one of the reagents is a biomolecule and the sensor is a biosensor. In certain other embodiments, the solid polymeric structure comprising pullulan and the reagents for performing a chemical reaction form a convenient device for performing a chemical reaction.

This application is a National Stage of co-pending InternationalApplication No. PCT/CA2014/051081 filed Nov. 10, 2014, which claims thebenefit of priority of U.S. provisional patent application No.61/901,784, filed on Nov. 8, 2013, the contents of both of which areincorporated herein by reference in their entirety.

FIELD

The present application relates to methods for performing chemicalreactions. In particular, the application is directed to methods inwhich two or more of the reagents for the chemical reaction arestabilized using water soluble polymers.

BACKGROUND

Almost all bioassays make use of bioreagents (such as enzymes andsmall-molecule substrates) that are labile to various degrees andrequire special shipping and storage. The instability of these moleculescan arise from either thermal denaturation or chemical modification,such as oxidation or hydrolysis. Because of these issues, they oftenhave to be shipped on dry ice with special packaging, which is costly.These reagents also have to be stored in bulk in refrigerators orfreezers to minimize loss of activity, but they must be retrieved,thawed, and aliquoted for intended tests that are often performed atroom temperature. Repeated freezing and thawing can result insignificant loss of activity, which often leads to less reliable testresults.

Pullulan is a natural polysaccharide produced by the fungusAureobasidium pullulans. ¹ It readily dissolves in water butresolidifies into films upon drying.^(1a,1b,2) The film forming propertyof pullulan has been utilized in some unique applications in thepharmaceutical and food industries, such as breath fresheners and foodadditives.^(1b,3) Recent studies have found that pullulan coatingsapplied to food packaging can act as oxygen barriers to prolong theshelf life of various foods.^(2,4) In addition, pullulan has been shownto preserve the viability of bacteria under various storage conditions.³

U.S. Pat. No. 7,604,807 describes the reversible preservation ofbiological samples in compositions comprising natural polymers such aspullullan or acacia gum.

SUMMARY

It has been discovered that unstable biomolecules, such as enzymes, andother unstable organic molecules can be stabilized for long periods oftime under ambient conditions when added to a pullulan solution andcasted as, for example, a film or pill. Pullulan is a non-ioniccarbohydrate, which is approved as a food additive and deemed safe.Addition of water or buffer to a pullulan-doped solid polymericstructure generates a solution with active molecules. This discovery maybe used to circumvent the need of cold condition for storage anddistribution for a variety of labile agents such as vaccines, andenzymes and reagents for performing chemical reactions. It was alsofound that because pullulan films are impermeable to oxygen, they can beused to preserve molecules that are easily oxidized.

In particular aspects of the present application the incorporation ofunstable molecules into pullulan has been found to facilitate andsimplify chemical reactions with these molecules. The molecules areconveniently packaged in pullulan polymeric structures andstored/packaged until the chemical reaction is needed or desired. Thisfinding is advantageous, for example, for the development of sensorsthat comprises sensitive biomolecules.

By placing reagents for a chemical reaction, for example reagents toperform an assay, into a pullulan polymeric structure, both premeasuredquantities of reagents and addition of preservatives that can prolongthe shelf life of the reagents are provided. It has been found hereinthat pullulan meets the following three conditions that are desirablefor this application: 1) it allows the encapsulation of molecules,including biomolecules, in a form suitable for shipping; 2) it providesoutstanding protection for entrapped molecules against thermaldenaturation and chemical modification during shipping and storage; and3) it is readily soluble in aqueous solution, allows the release of theencapsulated molecules, and does not interfere with the reaction(s)itself.

Accordingly, the present application includes a method of performing asingle step or multi-step chemical reaction comprising:

-   -   a) combining two or more reagents for the reaction, either        separately or together, with an aqueous pullulan solution to        provide reagent pullulan solutions or a reagent pullulan        solution, respectively;    -   b) drying the reagent pullulan solutions or the reagent pullulan        solution to provide solid polymeric structures or a solid        polymeric structure, respectively; and    -   c) if the two or more reagents are in separate solid polymeric        structures in b), then treating the solid polymeric structures        under conditions to dissolve the solid polymeric structures and        for the reagents to interact in a chemical reaction; or    -   d) if the two or more reagents are together in the solid        polymeric structure in b), then treating the solid polymeric        structure under conditions to dissolve the solid polymeric        structure and for the reagents to interact in a chemical        reaction.

The present application also includes sensors or devices comprising twoor more reagents entrapped in the same solid polymeric structure or indifferent solid polymeric structures wherein the solid polymericstructure(s) are comprised of pullulan. In an embodiment, at least oneof the two or more reagents is a biomolecule and the sensor is abiosensor.

In certain embodiments, the materials that are immobilized are inorganicor organic molecules, proteins, enzymes, antibodies, RNA, DNA, phage,viruses, anaerobic and aerobic bacteria, mammalian cells, vaccines or acombination thereof.

In other embodiments, the application includes a biosensor comprisingone or more water soluble films containing an active ingredient wherebyaddition of water or a buffer brings the active components in contactwith each other.

Other features and advantages of the present application will becomeapparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating embodiments of the application, are given byway of illustration only and the scope of the claims should not belimited by these embodiments, but should be given the broadestinterpretation consistent with the description as a whole.

DRAWINGS

The embodiments of the application will now be described in greaterdetail with reference to the attached drawings in which:

FIG. 1A shows approach I for a chemical reaction, in an exemplaryembodiment of the application, wherein at least two of the reagents arein separate solid polymeric structures (enzyme as a film, substrate as apill) includes the preparation and testing methods: a) preparation ofthe enzyme film: enzyme/pullulan solution is casted in an Eppendorftube, and left open to dry. b) preparation of substrate pill:substrate/pullulan solution is casted in mold with wells size of: 3 mmdiameter×3 mm depth. c) pesticide detection test.

FIG. 1B shows approach II for a chemical reaction, in an exemplaryembodiment of the application, wherein at least two of the reagents arein separate solid polymeric structures: (a) demonstration of the methodof constructing a capsule. For the purposes of nested capsules,different sizes are used. The liquid medium that the AChE and IDA isdissolved in should not be able to dissolve pullulan (ethanol/methanolfor the IDA, Tris for the AChE) (b) demonstrates a suggested method ofoperation for the capsule sensor. It is desirable to have either a smallvolume of sample for qualitative analysis, or a small, precisely knownvolume of sample for quantitative analysis, which is why it is suggestedto utilize a standard container that will result in consistent samplevolumes.

FIG. 2 shows (a) images of exemplary cast pullulan capsules. Capsulesare formed from 200 μL of 100 mg/mL pullulan solution, thereforeapproximately 20 mg of pullulan is used per capsule. (b) Schematicdiagram of each exemplary capsule's dimensions, as well as the mold thatthe capsules are cast into.

FIG. 3 shows images of the different sized capsules in exemplaryembodiments of the application. All capsules are cast from 200 mg/mLpullulan solution, and are ˜4 mm tall. The diameters are 2, 3, 4 mmrespectively.

FIG. 4 shows schematic diagrams of proposed capsule molds in exemplaryembodiments of the application. (a) demonstrates an angled entrance tothe mold, which should assist in the removal of the capsule, as well asprovide a large surface to assist in sealing the capsule with a film.(b) represents a proposed mold design in which the mold can be splitinto two parts, in order to allow for very easy removal of the capsule.Some clamping mechanism holds the two halves together when allowing thefilms to dry.

FIG. 5 shows a schematic diagram of a proposed exemplary paperlesspesticide sensor relying on the enzyme (AChE) and substrate (IDA), aswell as a side view of one of the proposed exemplary capsules and methodof sealing.

FIG. 6 shows the bluish green color change (dark) exhibited after 24hours of oxygen exposure in an exemplary sensor of the application. 5 μLof AChE, 5 μL of IDA and 35 μL of dH₂O were added. The AChE was castinto the bottom of the tube in 50 μL of a solution of 125 mg/mLpullulan.

FIG. 7 shows (a) an exemplary pullulan capsule containing a red dyepowder, used to determine if the capsule leaks, while (b) and (c)represents the color change that results when a single exemplary capsulecontaining 5 uL of IDA is added with 35 uL of water to the Eppendorftube containing 50 uL of 250 mg/mL pullulan and 10 uL of AChE enzyme.While difficult to see due to the picture quality, there is a distinctblue color change indicating activity of both the enzyme and thesubstrate.

FIG. 8 shows images of color intensity of the reaction over time in anexemplary sensor of the application. As can be seen, the reaction beginsto approach completion at time 15 minutes, and the color intensitychange is lessened. To generate the color, 5 μL of AChE was added to 40μL of 250 mg/mL pullulan solution, and cast into a 0.6 mL Eppendorfmicrocentrifuge tube. A 2 mm (diam)×4 mm (depth) capsule with anenlarged flange was filled with 5 μL of iDA (cap thickness of 5 mL, diamof 5/32″), and added to the Eppendorf tube 5 days after casting. 30 μLof dH₂O was added to the tube, to dissolve the capsule and allow for thereaction to occur.

FIG. 9 shows images of a sample with dH₂O, and a control sample testedwith no enzyme present in an exemplary sensor of the application. Thecontrol sample exhibited a slight color change after 60 minutes, butremained clear until 15 minutes, which is the typical sensing time.

FIG. 10 is a plot of the reaction rate as a function of the volume ofIDA added to the Eppendorf tube in an exemplary sensor of theapplication. It is expected that a higher reaction rate would result ina more intense color change in the allotted 15 minute time interval.

FIG. 11 shows plots of the absorbance measured for each sample solutionas a function of the concentration of pesticide added to the sample inan exemplary sensor of the application. A wavelength of 605 was used toensure that the correct trend is observed.

FIG. 12 shows images of the different well-patterns used in exemplarysensors of the application. When the pattern illustrated in (a) wasused, it was determined that the center well usually resulted in ahigher absorbance reading, most likely due to some sort of shadowingeffect. Accordingly, the wells were spaced out as illustrated in (b),which allows for a total of 30 samples to be analysed each time.

FIG. 13 is a plot of the absorbance as a function of time for a 0.5 mMIDA solution from an exemplary sensor of the application. The absorbanceplateaus as the reaction nears completion, at about 30 minutes.

FIG. 14 is a plot of the absorbance as a function of time for the 2.5 mMcase using an exemplary sensor of the application. The initial point of(0,00.03) is determined by a blank sample. The time delay before thestart of measurements represents the setup time from when the IDA isadded to when the plate is read in the Tecan. 1 uL of AChE (250 U/mL),194 uL Tris buffer and 5 uL of IDA in MeOH brings the total well volumeto 200 uL.

FIG. 15 shows plots of the reaction rate (abs*s−1) as a function of theIDA concentration from 0 to 4 mM using an exemplary sensor of theapplication. Plots are constructed with Sigma software, with the errorbars representing three standard deviations based on triplicate repeats.The kM and the vmax values agree between plots. (a) represents the 0-500s case, while (b) represents the 0-1500 s case.

FIG. 16 is a plot of absorbance vs. time for the three trials using anexemplary sensor of the application. It is evident that the reactionrate observed was faster for the fresh pullulan than the 10 day oldpullulan. Additionally, the initial slope of the solution with nopullulan was higher than that with pullulan. However, the final value ofthe solution without pullulan is both lower than the absorbance of thesolutions with pullulan.

FIG. 17 is a plot of the corrected reaction rate (abs*s−1) over timeusing an exemplary sensor of the application. The activity decreased toa mean of approximately 63% activity as compared to a fresh sample. Thisis an acceptable decrease in activity over 30 days, especially when itis considered that when AChE without any pullulan is left out for 24hours, it is completely inactivated (the rate of reaction is equal tothe rate of auto-hydrolyzation of IDA, indicating complete inactivationof the enzyme).

FIG. 18 shows a plot of the reaction rate of the enzyme (AChE) as afunction of the pullulan concentration using an exemplary sensor of theapplication.

FIG. 19 shows a graph of the reaction rate (abs/s) of AChE and IDA as afunction of the IDA concentration from 0 to 4 mM using an exemplarysensor of the application. The reaction rate of AChE tablet withdifferent concentrations of IDA was monitored to establish an optimalIDA concentration for pesticide detection experiment. From the data, theconcentration of IDA for the pesticide was chosen to be 2 mM (which issignificantly larger than KM). The error bars represent the standarddeviations based on triplicate repeats.

FIG. 20 shows fluorescence intensity-based thermal unfolding curves forHSA-buffer solution, in HSA-pullulan solution and HSA-pullulan film.Changes in the intrinsic fluorescence from tryptophan (Trp) residueswithin proteins can be used to provide information on proteinconformational stability and unfolding.⁵ Therefore, steady-statefluorescence spectra were measured at various temperatures forHSA-buffer solution, HSA-pullulan solution and HSA-pullulan film (HSAwas chosen for this study because it contains a single Trp allowing forunambiguous investigation). The data shows that the intensity offluorescence for HSA-buffer and HSA-pullulan solution decreased by morethan 90% when the temperature was raised from 20° C. to 90° C., whilethat of HSA-pullulan film only decreased by ˜70%. The unfoldingtemperature (wherein the Trp intensity is reduced by 50%) wassignificantly higher for the pullulan-HSA film at ˜80° C., versus ˜60°C. for both HSA-buffer and HSA-pullulan solutions. While not wishing tobe limited by theory, this study demonstrates that pullulan filmsignificantly enhances the thermal stability by preventing substantialunfolding as a result of molecular confinement in the rigid matrix. Thisstabilizing effect is only observed in the pullulan film and not in thepullulan solution.

FIG. 21 is a graph showing dose-dependent inhibition of AChE at varyingconcentrations of malathion using an exemplary sensor of theapplication.

FIG. 22 shows the evaluation of the long-term stability of AChE and IDAtablets stored at room temperature in exemplary embodiments of theapplication. Normalized activity of encapsulated AChE in reaction withfresh IDA, encapsulated IDA in reaction with fresh AChE, unencapsulated(native) AChE in reaction with fresh substrate, and unencapsulated IDAin reaction with fresh AChE.

FIG. 23 shows loss of AChE activity as a function of temperature inexemplary embodiments of the application. Both encapsulated AChE andnative enzyme were assessed.

FIG. 24 is a schematic showing the pill assay for ATP as an exemplarysensor of the application. (a) Firefly luciferase reaction. (b)Operation principle of the assay using ‘all-in-one’ pill.

FIG. 25 shows A) Glow kinetics of the exemplary ‘all-in-one’ pill afterone week of preparation and stored at room temperature; B) Stability ofthe pills over three weeks. The error bar represents triplicatemeasurements at each time interval.

FIG. 26 shows detection of ATP is solution using the exemplary‘all-in-one’ pills via a plot of the light intensity versus ATPconcentration ranging from 10 pM to 1000 pM in tricine buffer. The errorbars represent triplicate measurements at each concentration.

FIG. 27 shows detection of lysed (darker circles) and intact (lightercircles) E. Coli cells using the ‘all-in-one’ luminescent pill as anexemplary sensor of the application.

FIG. 28 shows the creation of exemplary pullulan films in (A)disc-shapes which are created by pipetting the pullulan mixture onsurface of the flexible transparency PET sheet, and (B) other desiredshapes and dimension.

FIG. 29 shows the release kinetics for an exemplary pullulan film. Zeroorder kinetics are shown by the total mass of Allura Red release as afunction of time

FIG. 30 shows the release kinetics for different exemplary polymericfilms.

FIG. 31 shows (A) a schematic of the reactions for Simon's assay todetect secondary amines where SNP is sodium nitroprusside; (B) sschematic of the lateral flow assay for the detection of secondaryamines with reagents required for the reaction immobilized in twodifferent pullulan films. The presence of a secondary amine in thesample results in the development of a strong cobalt-blue color, and (C)a schematic of the reformatting of Simon's assay as a spot-test(z-direction), by stacking the pullulan films containing the reagents,as an exemplary sensor of the application.

FIG. 32 shows a comparison of the color intensity for Simon's assay whenperformed using a lateral flow formatting (see FIG. 31B) and anexemplary z-directional format (see FIG. 31C). The quantities of allreagents used in both formats were the same.

FIG. 33 shows the formatting an E. coli detection assay in thez-direction as an exemplary sensor of the application that allows samplepreparation (cell lysis) and reporting in a without user intervention:(A) The basic components of the test comprise a pullulan film loadedwith a detergent (B-PER), lysozyme and DNase I for sample extraction anda paper disk containing CPRG (the substrate for β-galactosidase) and apoly-arginine layer; (B) details of films and reagents; (C) colorintensity obtained from the assay as a function of bacterial counts; and(D) multiple possibilities for formatting E. coli detection assay usingpaper disks, Eppendorf tube caps or in 96-well plates.

FIG. 34 shows agarose gel results of PCR products in the presence ofdifferent pullulan concentrations using exemplary sensors of theapplication.

DETAILED DESCRIPTION 1. Definitions

Unless otherwise indicated, the definitions and embodiments described inthis and other sections are intended to be applicable to all embodimentsand aspects of the present application herein described for which theyare suitable as would be understood by a person skilled in the art.

In understanding the scope of the present application, the term“comprising” and its derivatives, as used herein, are intended to beopen ended terms that specify the presence of the stated features,elements, components, groups, integers, and/or steps, but do not excludethe presence of other unstated features, elements, components, groups,integers and/or steps. The foregoing also applies to words havingsimilar meanings such as the terms, “including”, “having” and theirderivatives. The term “consisting” and its derivatives, as used herein,are intended to be closed terms that specify the presence of the statedfeatures, elements, components, groups, integers, and/or steps, butexclude the presence of other unstated features, elements, components,groups, integers and/or steps. The term “consisting essentially of”, asused herein, is intended to specify the presence of the stated features,elements, components, groups, integers, and/or steps as well as thosethat do not materially affect the basic and novel characteristic(s) offeatures, elements, components, groups, integers, and/or steps.

Terms of degree such as “substantially”, “about” and “approximately” asused herein mean a reasonable amount of deviation of the modified termsuch that the end result is not significantly changed. These terms ofdegree should be construed as including a deviation of at least ±5% ofthe modified term if this deviation would not negate the meaning of theword it modifies.

As used in this application, the singular forms “a”, “an” and “the”include plural references unless the content clearly dictates otherwise.For example, an embodiment including “a biomolecule” should beunderstood to present certain aspects with one biomolecule or two ormore additional biomolecules.

In embodiments comprising an “additional” or “second” component, such asan additional or second biomolecule, the second component as used hereinis chemically different from the other components or first component. A“third” component is different from the other, first, and secondcomponents, and further enumerated or “additional” components aresimilarly different.

The term “and/or” as used herein means that the listed items arepresent, or used, individually or in combination. In effect, this termmeans that “at least one of” or “one or more” of the listed items isused or present.

The term “chemical reaction” as used herein refers to any interactionbetween two or more molecules in which at least one of the molecules isaltered. Typically at least one of the altered molecules is referred toas a “product”.

The term “drying” as used herein refers to a process of allowing asolution of a polymer to cure or set until a solid, movable structure isobtained.

II. Methods of the Application

A method of performing a single step or multi-step chemical reactioncomprising:

-   -   a) combining two or more reagents for the reaction, either        separately or together, with an aqueous pullulan solution to        provide reagent pullulan solutions or a reagent pullulan        solution, respectively;    -   b) drying the reagent pullulan solutions or the reagent pullulan        solution to provide solid polymeric structures or a solid        polymeric structure, respectively; and    -   c) if the two or more reagents are in separate solid polymeric        structures in b), then treating the solid polymeric structures        under conditions to dissolve the solid polymeric structures and        for the reagents to interact in a chemical reaction; or    -   d) if the two or more reagents are together in the solid        polymeric structure in b), then treating the solid polymeric        structure under conditions to dissolve the solid polymeric        structure and for the reagents to interact in a chemical        reaction.

In an embodiment of the application at least one of the reagents is abiomolecule. In an embodiment, the biomolecule is selected from one ormore of a protein, enzyme, antibody, peptide, nucleic acid, phage,antidote and vaccine.

In an embodiment, at least one of the reagents is an enzyme. In anembodiment, the enzyme is selected from DNA polymerases, restrictionenzymes, DNA ligases, RNA ligases, luciferase, DNases, RNases,acetylcholine esterase, β-glucuronidase, β-galactosidase and lactatedehydrogenase. In an embodiment, the enzyme is selected from Taq DNApolymerase, phi29 DNA polymerase, Bst DNA polymerase, acetylcholineesterase, β-galactosidase, β-glucuronidase, luciferase and DNase.

In an embodiment, the nucleic acid is selected from single stranded DNA,double-stranded DNA, single-stranded RNA, double-stranded RNA, andchemically modified nucleic acid analogs in either single-stranded ordouble-stranded form. In an embodiment, the nucleic acid is selectedfrom DNA aptamers, RNA aptamers, riboswitches, DNAzymes, ribozymes,SOMOmers and Spiegelmers.

In an embodiment of the application at least one of the reagents iscomprised in a microorganism. In an embodiment, the microorganism isselected from one or more of anaerobic bacteria, aerobic bacteria,mammalian cells, bacterial cells and viruses.

In an embodiment of the application, at least one of the reagents is aninorganic or an organic molecule. In an embodiment, the inorganicmolecule is selected from one or more of an inorganic acid, an inorganicbase, an alkaline earth metal carbonate, an alkaline earth metalsulfate, an alkali metal carbonate, an alkali metal sulfate and a metalcomplex. In an embodiment, the inorganic molecule is a salt used inbutter solutions. In an embodiment, the organic molecule is selectedfrom one or more of indoxyl acetate, luciferin, lactate, acetaldehyde,chelating agents, NTPs (ATP, GTP, UTP and CTP) and dNTPs (dATP, dGTP,dTTP and dCTP).

In an embodiment the reagents are selected from substances used in cellgrowth media, for example, but not limited to a carbon source (such asglucose), salts needed for cell growth, a source of amino acids and asource of nitrogen. A person skilled in the art would appreciate thatthe components of a cell growth medium will vary depending on the celltype and the particular application, but would, none-the-less, be ableto select suitable components based on general knowledge in the art.

In an embodiment of the application, the conditions to dissolve thesolid polymeric structure(s) comprise contacting the solid polymericstructure(s) with water or an aqueous buffer. In an embodiment, thebuffer comprises tris(hydroxymethyl)aminomethane (Tris) and/orN-(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)glycine (Tricine). In a furtherembodiment, the conditions to dissolve the solid polymeric structurefurther allow the reagents to interact, via contact, in a chemicalreaction.

In an embodiment of the application, the two or more reagents are inseparate solid polymeric structures and the solid polymeric structuresare stacked in layers on top of each other. In an embodiment, the layersare stacked in an order that corresponds to an order required to performthe chemical reaction. For example, in a multi-step chemical reaction,the reagents that interact first would be comprised in the first or toplayer and/or in a solution being added to the first or top layer.Dissolution of the top polymeric structure layer results in a contactingof the reagents that interact first to provide a first reaction product.Once sufficient time has passed for the first reaction product to form,dissolution of a second pullulan layer comprising a third reagent occursunder conditions for the first reaction product to react with the thirdreagent to provide a second reaction product. This process can berepeated as many times as necessary to perform the entire multistepchemical reaction.

In an alternate embodiment, the two or more reagents are in separatesolid polymeric structures and the solid polymeric structures are in theshape of a pill, tablet or capsule, with each separate polymericstructure forming separate layers surrounding each other in the pill. Inthis embodiment, the reagents that interact first would be comprised inthe first or outer layer of the pill, tablet or capsule and/or in asolution being added to the first or outer layer. Dissolution of theouter polymeric structure layer results in a contacting of the reagentsthat interact first to provide a first reaction product. Once sufficienttime has passed for the first reaction product to form, dissolution of asecond pullulan layer, located internal to the outer pullulan layer andcomprising a third reagent, occurs under conditions the first reactionproduct to react with the third reagent to provide a second reactionproduct. This process can be repeated as many times as necessary toperform the entire multistep chemical reaction. Accordingly, in thisembodiment of the application, the layer on the outside of the pill,tablet or capsule comprises reagents that must react first in thechemical reaction and the remaining layers are arranged inside theoutside layer in an order that corresponds to an order required toperform the chemical reaction.

In an embodiment, the two or more reagents are in separate polymericstructures and the two or more reagents that are in separate polymericstructures comprise an enzyme or a receptor and a substrate for theenzyme or the receptor. In a further embodiment, dissolving a firstpolymeric structure in the presence of a test sample that is suspectedof comprising a modulator of the receptor or enzyme, followed bydissolution of a second polymeric structure comprising a substrate forthe enzyme or receptor is used in a method of assaying for modulatorsof, or molecules that bind to, the enzyme or receptor. In thisembodiment, if the final product of the chemical reaction differs in thepresence of the test sample compared to in the presence of a control(i.e. a sample comprising no suspected modulator or binder) then thetest sample comprises a modulator of, or a molecule that binds to, thereceptor or enzyme. In an embodiment, the final product of the chemicalreaction in the presence of the test sample or the final product of thechemical reaction in the absence of the test sample is detectable usingany known method. For example, the product is detectable usingcolorimetry, optical spectrometry and/or fluorescence.

Non-limiting examples of known enzymes and their known substrates anddetection systems that are amenable to adaptation and use in the methodof the present application are as follows:

-   -   (i) Acetylcholine        esterase—acetylthiocholine/dithiobisnitrobenzoate (DTNB);    -   (ii) Acetylcholine esterase—indophenyl acetate or indoxyl        acetate;    -   (iii) urokinase plasminogin activator (uPA)—S-2244;    -   (iv) adenosine triphosphatases (ATPases)/kinases—ATP-βS/DTNB;    -   (v) β-glucuronidase—5-bromo-4-chloro-3-indolyl-β-D-glucuronide        (X-GLUC)/FeCl₃/indigo dye;    -   (vi) β-galactosidase—bromo-chloro-indolyl-galactopyranoside        (X-GAL)/indigo dye    -   (vii) DNA/RNA/PNA aptamers, DNA/RNA enzymes or a DNA or RNA        aptazyme/signaling method;    -   (viii) functional nucleic acid/Φ29 DNA polymerase/Bst DNA        polymerase, circular template and dNTPs/gold nanoparticle        labeled linear DNA of the same sequence as the circular template        (or a portion thereof).

In an embodiment, the two or more reagents that are in separatepolymeric structures comprise AChE and indoxyl acetate.

In an embodiment, the two or more reagents that are in separatepolymeric structures comprise (1) reagents for cell lysis and (2) asubstrate for an enzyme that is released from a cell in the presence ofthe reagents for cell lysis. In an embodiment, the reagents for celllysis are selected from one or more of detergents, lysozyme, lyticbacteriophage and DNase I. In a further embodiment, the enzyme that isreleased from a cell in the presence of the reagents for cell lysis isβ-galactosidase or β-glucuronidase. In a further embodiment, thesubstrate for the enzyme is selected from5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-GLUC),bromo-chloro-indolyl-galactopyranoside (X-GAL), 5-bromo-3-indolylβ-D-galactopyranoside (Bluo-Gla), 5-bromo-6-chloro-3-indolylβ-D-galactopryaniside (Magenta-Gal), 6-chloro-3-indolylβ-D-galactopyranoside (Salmon-Gal), 2-nitrophenyl β-D-galactopyranoside(ONPG) and 4-nitro β-D-galactopyranoside (PNPG).

In an embodiment, the two or more reagents that are in separatepolymeric structures comprise the reagents to perform the Simon's testfor secondary amines. In this embodiment, the top or first layer in astacked sensor, or the first or outer layer of a pill, tablet orcapsule, comprises acetaldehyde and the second or inner layer comprisessodium nitroprusside and sodium carbonate. To perform the method of theapplication, the top layer of a stack of solid polymeric structures orthe outer layer of a pill, tablet or capsule is contacted with anaqueous solution that is suspected of comprising a secondary amine. Ifthe blue color of the Simon-Awe complex is detected, then the samplecomprised a secondary amine.

It is an embodiment of the application that timing of dissolution of thelayers, whether stacked or in a pill, tablet or capsule, is controlledto allow sufficient reaction time for each step of the chemicalreaction. In an embodiment, the timing is controlled by one or more ofthickness of the layers and pullulan concentration.

In an embodiment of the application, the two or more reagents are in thesame solid polymeric structure. In this embodiment, the solid polymericstructure is optionally referred to as an “all-in-one” pill, tabletcapsule or film.

In an embodiment, the two or more reagents in the same solid polymericstructure comprise reagents for detection of adenosine triphosphate(ATP). In an embodiment the reagents for ATP detection compriseluciferin, luciferase, coenzyme A (CoA), dithiothreitol (DTT), achelating reagent, MgCO₃ and MgSO₄. In an embodiment, the chelatingagent is ethylenediaminetetraacetic acid (EDTA).

In another embodiment, the two or more reagents in the same solidpolymeric structure comprise reagents for rolling circular amplification(RCA). In a embodiment, the reagents for RCA comprise DNA polymerase(such as phi29 DNA polymerase), circular template DNA and buffer salts.

In another embodiment, the two or more reagents in the same solidpolymeric structure comprise reagents for cell growth media. In anembodiment, reagents for cell growth media comprise carbon source (suchas glucose), salts needed for cell growth, a source of amino acids and asource of nitrogen.

In another embodiment, the two or more reagents in the same solidpolymeric structure comprise reagents for PCR. In an embodiment, thereagents for PCR comprise DNA polymerase (for example Taq DNApolymerase_, linear template DNA and buffer salts.

In the above “all-in-one” embodiments, the present application providesthe advantage of having many or all of the reagents required for achemical reaction, such as, but not limited to, cell growth, nucleicacid amplification, and ATP detection, in a single stable solidpolymeric structure that is readily stored and transported. The reagentsare optionally, premeasured and/or stabilizing reagents, such asantibiotics, are added.

In an embodiment of the application, the two or more reagents are inseparate solid polymeric structures and the solid polymeric structuresare each dissolved in a single water or buffer solution releasing thereagents for interaction in the chemical reaction. In this embodiment,it is a further embodiment that the solid polymeric structures are,independently, in a shape selected from a pill, tablet and capsule.

It is an embodiment that the amount of the two or more reagents that arecombined with the aqueous pullulan solution is premeasured. In anembodiment, the premeasured amount corresponds to amounts and ratios ofreagents needed to perform the chemical reaction(s). In a furtherembodiment, stabilizing agents, such as antimicrobial agents, areincluded in the aqueous pullulan solution.

In an embodiment, the chemical reaction produces a product that isdetectable using any known method. For example, the product isdetectable using colorimetry, optical spectrometry and/or fluorescence.

In an embodiment, the concentration of the aqueous pullulan solutions isabout 5% (w/v) to about 25% (w/v). In a further embodiment, theconcentration of the aqueous pullulan solutions is about 100/(w/v) toabout 20% (w/v). In a further embodiment, the concentration of theaqueous pullulan solutions is about 11% (w/v) to about 13% (w/v), orabout 12% (w/v).

In an embodiment of the application the one or more of solid polymericstructures is cast onto a substrate comprising at least one reagent forthe chemical reaction. In this embodiment, dissolution of the solidpolymeric structure on the substrate brings the reagent(s) in the solidpolymeric structure into contact with the reagent(s) on and/or in thesubstrate to interact in a chemical reaction. In an embodiment, thesubstrate is a paper-based substrate. In a further embodiment, thesubstrate is surface treated with the at least one reagent for thereaction.

III. Sensors and Devices of the Application

The present application also includes sensors or devices comprising twoor more reagents entrapped in the same solid polymeric structure or indifferent solid polymeric structures wherein the solid polymericstructure(s) are comprised of pullulan. In an embodiment, at least oneof the two or more reagents is a biomolecule and the sensor is abiosensor.

In an embodiment, the biomolecule is selected from one or more of aprotein, enzyme, antibody, peptide, nucleic acid, phage, antidote andvaccine. In an embodiment, at least one of the reagents is an enzyme. Inan embodiment, the enzyme is selected from DNA polymerases, restrictionenzymes, DNA ligases, RNA ligases, luciferase, DNases, RNases,acetylcholine esterase, β-glucuronidase, β-galactosidase and lactatedehydrogenase. In an embodiment, the enzyme is selected from Taq DNApolymerase, Φ29 DNA polymerase, Bst DNA polymerase, acetylcholineesterase, β-galactosidase, β-glucuronidase, luciferase and DNase. In anembodiment, the nucleic acid is selected from single stranded DNA,double-stranded DNA, single-stranded RNA, double-stranded RNA, andchemically modified nucleic acid analogs in either single-stranded ordouble-stranded form. In an embodiment, the nucleic acid is selectedfrom DNA aptamers, RNA aptamers, riboswitches, DNAzymes, ribozymes,SOMOmers and Spiegelmers.

In an embodiment of the application at least one of the reagents iscomprised in a microorganism. In an embodiment, the microorganism isselected from one or more of anaerobic bacteria, aerobic bacteria,mammalian cells, bacterial cells and viruses.

In an embodiment of the application, at least one of the reagents is aninorganic or an organic molecule. In an embodiment, the inorganicmolecule is selected from one or more of an inorganic acid, an inorganicbase, an alkaline earth metal carbonate, an alkaline earth metalsulfate, an alkali metal carbonate, an alkali metal sulfate and a metalcomplex. In an embodiment, the inorganic molecule is a salt used inbuffer solutions. In an embodiment, the organic molecule is selectedfrom one or more of indoxyl acetate, luciferin, lactate, acetaldehyde,chelating agents, NTPs (ATP, GTP, UTP and CTP) and dNTPs (dATP, dGTP,dTTP and dCTP).

In an embodiment the reagents are selected from substances used in cellgrowth media, for example, but not limited to a carbon source (such asglucose), salts needed for cell growth, a source of amino acids and asource of nitrogen. A person skilled in the art would appreciate thatthe components of a cell growth medium will vary depending on the celltype and the particular application, but would, none-the-less, be ableto select suitable components based on general knowledge in the art.

In an embodiment of the application, the two or more reagents are inseparate solid polymeric structures and the solid polymeric structuresare stacked in layers on top of each other. In an embodiment, the layersare stacked in an order that corresponds to an order required to performthe chemical reaction. For example, in a multi-step chemical reaction,the reagents that interact first would be comprised in either the firstor top layer and/or in a solution being added to the first or top layer.

In an alternate embodiment, the two or more reagents are in separatesolid polymeric structures and the solid polymeric structures are in theshape of a pill, tablet or capsule, with each separate polymericstructure forming separate layers surrounding each other in the pill. Inthis embodiment, the reagents that interact first would be comprised ineither the first or outer layer of the pill, tablet or capsule and/or ina solution being added to the first or outer layer. Dissolution of theouter polymeric structure layer results in a contacting of the reagentsthat interact first to provide a first reaction product. Once sufficienttime has passed for the first reaction product to form, dissolution of asecond pullulan layer, located internal to the outer pullulan layer andcomprising a third reagent, occurs under conditions for the firstreaction product to react with the third reagent to provide a secondreaction product. Accordingly, in this embodiment of the application,the layer on the outside of the pill, tablet or capsule comprisesreagents that must react first in the chemical reaction and theremaining layers are arranged inside the outside layer in an order thatcorresponds to an order required to perform the chemical reaction.

In an embodiment, the two or more reagents are in separate polymericstructures and the two or more reagents that are in separate polymericstructures comprise an enzyme or a receptor and a substrate for theenzyme or the receptor. Non-limiting examples of known enzymes and theirknown substrates and detection systems that are amenable to adaptationand use in the method of the present application are as follows:

-   -   (i) Acetylcholine        esterase—acetylthiocholine/dithiobisnitrobenzoate (DTNB);    -   (ii) Acetylcholine esterase—indophenyl acetate or indoxyl        acetate;    -   (iii) urokinase plasminogin activator (uPA)—S-2244;    -   (iv) adenosine triphosphatases (ATPases)/kinases—ATP-βS/DTNB;    -   (v) β-glucuronidase—5-bromo-4-chloro-3-indolyl-β-D-glucuronide        (X-GLUC)/FeCl₃/indigo dye;    -   (vi) β-galactosidase—bromo-chloro-indolyl-galactopyranoside        (X-GAL)/indigo dye    -   (vii) DNA/RNA/PNA aptamers, DNA/RNA enzymes or a DNA or RNA        aptamzyme/signaling method;    -   (viii) functional nucleic acid/Φ29 DNA polymerase/Bst DNA        polymerase, circular template and dNTPs/gold nanoparticle        labeled linear DNA of the same sequence as the circular template        (or a portion thereof).

In an embodiment, the two or more reagents that are in separatepolymeric structures comprise AChE and indoxyl acetate.

In an embodiment, the two or more reagents that are in separatepolymeric structures comprise (1) reagents for cell lysis and (2) asubstrate for an enzyme that is released from a cell in the presence ofthe reagents for cell lysis. In an embodiment, the reagents for celllysis are selected from one or more of detergents, lysozyme, lyticbacteriophage and DNaseI. In a further embodiment, the enzyme that isreleased from a cell in the presence of the reagents for cell lysis isβ-galactosidase or β-glucuronidase. In a further embodiment, thesubstrate for the enzyme is selected from5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-GLUC),bromo-chloro-indolyl-galactopyranoside (X-GAL), 5-bromo-3-indolylβ-D-galactopyranoside (Bluo-Gla), 5-bromo-6-chloro-3-indolylβ-D-galactopryaniside (Magenta-Gal), 6-chloro-3-indolylβ-D-galactopyranoside (Salmon-Gal), 2-nitrophenyl β-D-galactopyranoside(ONPG) and 4-nitro β-D-galactopyranoside (PNPG).

In an embodiment, the two or more reagents that are in separatepolymeric structures comprise the reagents to perform the Simon's testfor secondary amines. In the embodiment, the top or first layer in astacked sensor, or the first or outer layer of a pill, tablet or capsulecomprises acetaldehyde and the second or inner layer comprises sodiumnitroprusside and sodium carbonate.

It is an embodiment of the application that timing of dissolution of thelayers, whether stacked or in a pill, tablet or capsule, is controlledto allow sufficient reaction time for each step of the chemicalreaction. In an embodiment, the timing is controlled by one or more ofthickness of the layers and pullulan concentration.

In an embodiment of the application, the two or more reagents are in thesame solid polymeric structure. In this embodiment, the solid polymericstructure is optionally referred to as an “all-in-one” pill, tabletcapsule or film.

In an embodiment, the two or more reagents in the same solid polymericstructure comprise the reagents for detection of adenosine triphosphate(ATP). In an embodiment the reagents for ATP detection compriseluciferin, luciferase, coenzyme A (CoA), dithiothreitol (DTT), achelating reagent, MgCO₃ and MgSO₄.

In another embodiment, the two or more reagents in the same solidpolymeric structure comprise reagents for rolling circular amplification(RCA). In a embodiment, the reagents for RCA comprise DNA polymerase(such as phi29 DNA polymerase), circular template DNA and buffer salts.

In another embodiment, the two or more reagents in the same solidpolymeric structure comprise reagents for cell growth media. In anembodiment, reagents for cell growth media comprise carbon source (suchas glucose), salts needed for cell growth, a source of amino acids and asource of nitrogen.

In another embodiment, the two or more reagents in the same solidpolymeric structure comprise reagents for PCR. In an embodiment, thereagents for PCR comprise DNA polymerase (such as Taq DNA polymerase),linear template DNA and buffer salts.

In the above “all-in-one” embodiments, the present application providesthe advantage of having many or all of the reagents required for achemical reaction, such as, but not limited to, cell growth, nucleicacid amplification, and ATP detection, in a single stable solidpolymeric structure that is readily stored and transported. The reagentsare optionally, premeasured and/or stabilizing reagents, such asantibiotics, are added.

In an embodiment of the application, the two or more reagents are inseparate solid polymeric structures and the solid polymeric structuresare each dissolved in a single water or buffer solution releasing thereagents for interaction in the chemical reaction. In this embodiment,it is a further embodiment that the solid polymeric structures are,independently, in a shape selected from a pill, tablet and capsule.

It is an embodiment that the amount of the two or more reagents that arecombined with the aqueous pullulan solution are premeasured. In anembodiment, the premeasured amount corresponds to amounts and ratios ofreagents needed to perform the chemical reaction(s). In a furtherembodiment, stabilizing agents, such as antimicrobial agents, areincluded in the solid polymeric structure(s).

In an embodiment, the chemical reaction produces a product that isdetectable using any known method. For example, the product isdetectable using colorimetry, optical spectrometry and/or fluorescence.Accordingly, in an embodiment of the application the sensors comprise adetection device.

In an embodiment of the application the one or more of solid polymericstructures is cast onto a substrate comprising at least one reagent forthe chemical reaction. In this embodiment, dissolution of the solidpolymeric structure on the substrate brings the reagent(s) in the solidpolymeric structure into contact with the reagent(s) on and/or in thesubstrate to interact in a chemical reaction. In an embodiment, thesubstrate is a paper-based substrate. In a further embodiment, thesubstrate is surface treated with the at least one reagent for thereaction.

In an embodiment, the solid polymeric structures comprise about 5% (w/v)to about 25% (w/v) of the pullulan. In a further embodiment, the solidpolymeric structures comprise about 10% (w/v) to about 20% (w/v) of thepullulan. In a further embodiment, the solid polymeric structurescomprise about 11% (w/v) to about 13% (w/v), or about 12% (w/v), of thepullulan.

In an embodiment, the sensors and devices of the application arecomprised in commercial kits for performing the desired chemicalreaction(s).

The following are specific, non-limiting examples of some sensors anddevices of the present application:

(a) AChE Sensors

A sensor for the detection of inhibitors of acetylcholine esterase(AChE) comprising:

a) a pullulan polymeric structure comprising the AChE; and

b) a pullulan polymeric structure comprising indole acetic acid (IDA),

wherein a) and b) are arranged so that a) is first contacted with asample comprising one or more test substances under conditions todissolve the pullulan polymeric structure and for the one or more testsubstances and the AChE to interact to provide a first reaction solutionfollowed by contacting b) with the first reaction solution.

In an embodiment of the application, the pullulan polymeric structure ina) is a film on the inner wall of a reaction vessel and the pullulanpolymeric structure in b) is a pill, capsule or tablet.

In an embodiment of the application the sensor for the detection ofinhibitors of acetylcholine esterase is a layered pill, tablet orcapsule wherein the pullulan polymeric structure in a) forms an outerlayer and the pullulan polymeric structure in b) forms an inner layer.

In an embodiment, the outer and inner layers are adjacent and contactingeach other.

In an embodiment of the application, the pullulan polymeric structure ina) and the pullulan polymeric structure in b) are both films that arestacked in layers adjacent and contacting each other.

In an embodiment of the application, the timing of the dissolution ofthe layers is controlled to allow sufficient time for the one or moretest substances and the AChE to interact to provide a first reactionsolution. In an embodiment, the timing is controlled by one or more ofthickness of the layers and pullulan concentration.

In an embodiment, the pullulan polymeric structure in a) and thepullulan polymeric structure in b) are cast in two separate structuresthat are added sequentially to the sample comprising one or more testsubstances, with the pullulan polymeric structure in a) being added tothe sample prior to the pullulan polymeric structure in b), and whereinthe pullulan polymeric structure in b) is added to the sample when thepullulan polymeric structure in a) has dissolved and the one or moretest substances and the AChE have interacted to provide the firstreaction solution.

(b) β-Galactosidase Sensors

It has been demonstrated herein that stacked pullulan films with variousreagents can be used to perform multi-step reactions in a single step toproduce sensor outputs based on complex reaction sequences. In addition,such reactions can utilize labile and/or volatile reagents, as theseremain intact and immobilized when present in pullulan films. Thearrangement and thickness of the stacked films can be varied to controlreaction timing and sequence, or incorporate delays for samplepreparation or incubation prior to detection steps. This time dependentreagent deliverer allows the user to manipulate fluid movement in allthree directions (x, y and z directions) to create multi-step reactionsand assays. Using this technology, paper based analytical devices werecreated for the detection of secondary amines a methamphetamine mimic(DEA) and a fecal coliform (E. coli O157). These sensors avoidedpreviously required sample handling steps, such as cell lysis, and thussimplified the assays. Given the dual benefits of reagent stabilizationand controlled release, this format should be amenable to a wide rangeof complex sequential reactions on paper-based microanalytical devices.

Accordingly, the present application includes a sensor for the detectionof β-galactosidase activity from a microorganism comprising:

-   -   a) a pullulan polymeric film comprising a detergent, lysozyme        and DNase I; and    -   b) a substrate surface treated with chlorophenol red        β-D-galactopyranoside (CPRG), and optionally, polyarginine,        wherein a portion of b) is coated with the pullulan polymeric        film of a).

(c) Secondary Amine Sensors

The present application also includes a sensor for the detection ofsecondary amines comprising

-   -   a) a pullulan polymeric structure comprising acetaldehyde; and    -   b) a pullulan polymeric structure comprising sodium        nitroprusside and sodium carbonate,        wherein a) and b) are arranged so that a) is first contacted        with a sample comprising one or more test substances under        conditions to dissolve the pullulan polymeric structure and for        the one or more test substances and the acetaldehyde to interact        to provide a first reaction solution followed by contacting b)        with the first reaction solution.

In an embodiment of the application, the pullulan polymeric structure ina) and the pullulan polymeric structure in b) are both films that arestacked in layers adjacent and contacting each other.

In an embodiment of the application, the timing of the dissolution ofthe layers is controlled to allow sufficient time for the one or moretest substances and the acetaldehyde to interact to provide a firstreaction solution. In an embodiment, the timing is controlled by one ormore of thickness of the layers and pullulan concentration.

(d) ATP Detection

A highly strenuous and time-consuming luminescence assay was madesingle-step by encapsulating all the reagents, including highly unstableenzyme and substrate in a single pill using pullulan. All the componentswere thermally stable in the pills which provided sensitive ATPdetection up to picomolar concentration. This cost-effective method (˜88Canadian cents per 100 pills which is up to 130 times cheaper than thesolution-based commercial assay kits), is also easy to scale-up. Thisstabilization technique will cover a broad spectrum of applicationranging from inexpensive real-time ATP testing to high-throughputscreening applications where sensitivity and stable light emission aredesired.

Accordingly, the present application also includes a sensor for ATPdetection comprising all reagents for ATP detection in a pullulanpolymeric structure. In an embodiment, the reagents for ATP detectioncomprise luciferin, luciferase, coenzyme A (CoA), dithiothreitol (DTT),a chelating reagent, MgCO₃ and MgSO₄.

(e) Nucleic Acid Amplification

The present application also includes a device for amplifying DNA usingPCR comprising a pullulan polymeric structure comprising DNA polymerase(such as Taq DNA polymerase), linear template DNA and buffer salts. Thepresent application also includes a device for amplifying DNA using RCAcomprising a pullulan polymeric structure comprising DNA polymerase(such as phi29 DNA polymerase), linear template DNA and buffer salts. Inan embodiment, the device is in the form of a tablet, pill or capsule.

(f) Cell Growth Media

The present application also includes a device for transporting cellgrowth media comprising a pullulan polymeric structure comprisingreagents for cell growth media. In an embodiment, reagents for cellgrowth media comprise carbon source (such as glucose), salts needed forcell growth, a source of amino acids and a source of nitrogen.

EXAMPLES

The following non-limiting examples are illustrative of the presentapplication:

Example 1: Pullulan Encapsulated Enzyme/Substrate

It is suggested that, since paper is very non-uniform and cannot becounted on for reliable results, to remove the paper from a sensingsystem. This could be accomplished by creating pullulan capsules whichshows high oxygen barrier properties while it is a fast dissolvablepolymer. Here in two separate parts, are first explained the methods ofcreating capsules in different shape and sizes, then the idea ofcreating a lab on a capsule, particularly the pesticide detectionsensor, is explored. Two approaches are considered for creating thepesticide sensor (see FIGS. 1A and 1B): Approach I) Two separate pills:Pullulan/Enzyme film is casted inside of an Eppendorf tube, while IDA isseparately entrapped in a pullulan pill. In this case the sensoroperation method would be: First 200 μl of the water sample is added tothe Eppendorf tube which its inner wall is covered with previouslycasted AChE-pullulan, after around 5 minutes allowing incubation of thereleased enzyme, then the IDA-pullulan pill is dropped into the tube andlet it to be released and react with un-inhibited enzyme to developedcolor. Approach II) Unique capsule: the IDA-pullulan pill isencapsulated inside a AChE-pullulan capsule. So as the result, when thecapsule is dropped to the sample solution, it starts to dissolve theouter layer of the capsule which includes the enzyme (AChE) inapproximately 5 minutes allowing incubation of the released enzyme. Thesample solution then dissolves the inner capsule allowing the substrateto be released and react with un-inhibited enzyme to developed color.

(a) Pullulan Capsules

To make the capsules, 100 mg of pullulan was dissolved in 1 mL of dH₂O,and 200 μL of the resulting solution was added to a prepared mold (3capsules at a time). This mold was then allowed to dry for approximately22 hours, followed by 2 hours in the oven at about 75° C. to completethe drying process. The capsules were cast into the smaller holes in themold, while the larger holes were used to cast a large capsule, whichrequired 270 μL of the solution. FIG. 2 contains images of the completedcapsules.

Casting Capsules (Different Sizes)

Different size capsules (pullulan) were tested. These capsule sizeswere: 2 mm, 3 mm, 4 mm and 5 mm respectively (diameter). The depth ofall capsules was 4 mm. These molds were filled with pullulan solution of100 mg/mL, 200 mg/mL and 300 mg/mL respectively, and allowed to dryovernight. The resulting capsules revealed that the 100 mg/mL was toothin to be of practical use, and the 300 mg/mL was very difficult toremove from the mold. However, the 200 mg/mL returned very good capsulesas pictured in FIG. 3.

It was decided to cast capsules in different formats, so as to allow foreasy release of the capsules, as well as a capsule shape that moreeasily allows for sealing the top on. The methods investigated are shownin FIG. 4. Additionally, it was observed that if the capsule mold waswarmed up to 70° C. after the capsules were dried, the holes expand andallow for easy release of the capsule.

Sealing Capsules while in Mold

It is also suggested that it may be easier to seal the capsules insidethe mold, as the mold would hold the capsule in the right orientationand perhaps allow for an easier application of the film.

A modification of this idea was to seal the capsules after making acustom-sized hole in a Styrofoam block to support the capsule while itis sealed. This worked quite well.

Casting Under a Nitrogen Blanket

It is attempted to remedy the issue of poor enzyme maintenance bycasting the films under a nitrogen blanket, which should prevent enzymeoxidation as a result of recirculation during the casting procedure. Toserve as a control, a film is also cast in the bio safety cabinetsimultaneously, where it is in contact with oxygen during the castingprocedure. Another film is cast in a beaker containing a plastic petridish with a brass ring with a constant supply of nitrogen gas a lowpressure/flow rates.

Casting Pullulan Capsules

Several capsules were cast in which 75 μL of 300 mg/mL plain pullulansolution was added to the caps of small eppendorf (microcentrifuge)tubes. These capsules were then left for 2 days to allow for completedrying (in a fume hood).

In order to compare the functionality of pullulan vs. polyvinyl alcohol(PVA) in a capsular format, 10 capsules were also cast of PVA. Since PVAis a much denser solution, only 100 mg/mL solution was used.

PVA Capsule Casting and Testing

Some capsules were formed from PVA to test if it is a viable alternative(much cheaper) to pullulan. 75 μL of 150 mg/mL PVA was cast intoEppendorf microcentrifuge tube caps. The resulting capsules were foundto be very easy to remove from the mold, did not dissolve in ethanol,and were (slowly) soluble in water. These are desirable characteristicsof the polymer. A 5 mL film of PVA was also cast (3.5 cm Falcon petridish, 5 mL volume of 50 mg/mL PVA solution), and capsules were formed ona glass surface using the brass rings (microscope slide glass (smooth)and ½″ brass casting rings). The resulting capsules were then tested todetermine solubility. PVA capsules and films were colorless and flexible(more so than pullulan).

However, the PVA was much slower to dissolve to form a solution, andtypically required the application of heat (˜80° C.) on a magnetic stirplate. When the PVA solution was prepared using microfuge tubes and thevortex, a foam was created that did not go away, rendering the solutionuseless.

Several of the PVA capsules were used to determine if they wereapplicable to the encapsulation of smell enzymes, and tested with skunkoil dissolved in methanol. The methanol resulted in the capsule becomingvery flexible and it contracted, not sealing properly.

Dissolution of PVA Capsules

In order to test how long the PVA capsules take to release a dye,several milligrams of Allura Red dye (in powder form) was added to theinside of several 100 mg/mL PVA capsules, and the top was sealed with aPVA glue solution and a 5 mL (50 mg/mL) 3.5 cm cast film punched to5/32″. The sealed capsules were then washed with ethanol, and added to100 uL of dH₂O to determine how long it takes for the dye to bereleased. The results indicated that the release time is between 2 minand 4 min 10 sec. For a reliable inactivation of the AChE by anypesticide in a sample solution, a minimum incubation period of ˜6minutes is required, hence the PVA capsules (unless cast thicker) willnot function as a release mechanism.

Example 2: Pesticide Sensor Capsules

It is suggested that in a paper-based sensor, since the paper is verynon-uniform and cannot be counted on for reliable results, to remove thepaper from the sensing system. This could be accomplished by creatingpullulan capsules, as previously described, and nesting two capsulestogether, as demonstrated in FIG. 4.

As can be seen from FIG. 5, the outermost pullulan capsule layer willdissolve first, when contacted with an aqueous sample. The pullulancapsule should be thin enough to allow for rapid dissolution. If thereare any pesticides present in the water, the pesticides will rapidlyinactivate the enzyme. Approximately a 5 minute incubation period isdesirable. During this time, the inner capsule (containing IDA) shouldnot dissolve. Therefore, the capsule may be composed of:

-   -   i. a thicker layer of pullulan that will resist complete        dissolution for about 5 minutes    -   ii. a thinner layer of another, slower-to-dissolve polymer that        can be cast into a capsule shape using the same method.

The basic construction process for the capsules has already beenintroduced, as demonstrated in FIG. 1B. In order to create ‘nested’capsules, two capsules of different sizes are cast.

An issue with this type of sensor is the sample size. If a very largevolume is used, the AChE enzyme will dissipate, especially if someconvective currents occur, or non-stagnant sample is used. In this case,by the time the 5 minute capsule inside has dissolved, the IDA will notcome into contact with enough AChE in a small enough area to result inany visible color change.

It is suggested to use a sample container which possesses a known volume(relatively small). The resultant color intensity of the liquid sampleafter a certain time frame (should be ˜6 minutes) can be used to form astandard curve against pesticide concentration, and the device should beable to be used as a quantifiable sensor. For a qualitative sensor only,any blue color change should be clear enough to identify in a smallvolume, particularly if the color of the container is selected so as tomaximize the contrast between any blue color and the background color.

As a starting point, the film or capsules that encapsulate the IDA andthe enzyme-containing films as well are created, as a proof of concept.

The films that are used to encapsulate the IDA were formed by preparinga solution of 7 mL of dH₂O and 350 mg of pullulan powder (dissolvedcompletely) and cast into a 3.5 cm Falcon petri dish. Then, ½″ circularfilms were punched out of it, and a thick pullulan solution (used as aglue) was added to the films around the outside to stick them together(with the IDA solution/powder in the centre).

The films that were used to encapsulate the enzyme (AChE) were formed bymixing 0.9217 mL of dH₂O with 46.083 mg of pullulan, and adding therequired amount of AChE for each film. Three (3) films with 5 μL of AChEand two (2) films with 10 μL of AChE were tested, in order to determinewhich volume of enzyme results in a better color change.

In order to cast the films with enzyme, to avoid wasting the enzyme, thefilms were cast into brass or Teflon rings, on a glass petri dish. Itwas observed that brass rings on a glass dish or Teflon rings on aplastic dish allowed the solution to leak out underneath the ring,ruining results. Teflon rings on a glass surface and brass rings on aplastic surface were therefore used when casting films.

Capsules Cast in a 96 Well Plate

In order to determine if pullulan capsules can be cast into the 96 wellplates, which would allow for automated analysis of color intensity in a96 well plate, it was attempted to cast some films into several wells.When cast into the wells, different concentrations of the pullulansolution were used, ranging from 50 mg/mL to 350 mg/mL pullulan. Theresulting capsules were then checked upon drying, and it was found thatthe PEG-350 ingredient in the solution resulted in holes being formed inthe capsules. Nonetheless, each capsule was formed in the correct shape,although the 50 and 100 mg/mL concentration capsules were too thin to beuseful. 400 μL of pullulan solution was added to each well.

Enzyme Activity Testing

In order to determine if the casting method reliably worked forimmobilizing enzymes within the pullulan solution, the portions of thefilm that leaked out of the brass rings onto the plastic petri disheswere collected and tested. Three samples were tested:

5 mg/mL AChE, ˜10 mg film, 50 μL dH₂O, 2 mg IDA→slight greenish bluecolor change, but not nearly noticeable enough to be satisfactory.

10 mg/mL AChE, ˜10 mg film, 350 μL dH₂O, 5 mg IDA→too much water presentfor any detectable color change.

10 mg/mL AChE, ˜10 mg film, 50 μL dH₂O, 10 mg IDA→slightly greenishcolor change, no blue. Color changed to yellow overnight for reasonsunknown (most likely excess IDA)

The films that were cast with the enzyme (the amount that did not leakfrom under the brass rings) were tested simultaneously with IDA, but nocolor change was visible.

It is hypothesized that the lack of enzyme activity was due to either(a) the film being too thin, resulting in poor protection of the enzyme,or (b) while drying, too much of the enzyme came into contact withoxygen due to recirculation within the pullulan solution.

Best Concentrations for Color Change

To determine the ideal concentration (of enzyme and pullulan) thatallows for a good color change that will noticeably diminish withpesticide presence, several trials were conducted with differentconcentrations. Considered were pullulan concentrations of 125 mg/mL and250 mg/mL and AChE volumes of 5 μL and 10 μL respectively. Four sampleswere prepared by mixing the pullulan and water together, to form 40 μL,then the enzyme was added and the total volume was set to 50 μL (by theaddition of water). 2 mg of IDA was added to each tube.

Pullulan Water AChE Tube (mg/mL) (μL) (μL) 1 125 5 5 2 125 0 10 3 250 55 4 250 0 10

It was observed that in the short term, the color intensities developedin tubes 1 and 2 (lower concentrations of pullulan) were darker, but inlong term, the intensities of 1 and 3, and 2 and 4 respectively, matchedvery closely. This indicates that: (a) the volume of enzyme is directlyrelated to the intensity of color change, ie. the more enzyme ispresent, the more intense the color change will be, and (b) a higherconcentration of pullulan results in the reaction occurring slower, butthe end result (intensity) is the same, it just takes longer to reachit.

It may not always be the case that the most intense color change is themost desirable, as it is more difficult for low concentrations ofpesticide to diminish the color noticeably, so this could result in somefalse negative results if the color change is too intense. In order totest if the color change will be approximately the same when the samesolutions are dried, four eppendorf tubes were prepared with solutionsmatching those in tubes 1 and 4 from the previous results.

Tube ID Pullulan (mg/mL) Enzyme (μL) 1-1 125 5 1-2 125 5 4-1 250 10 4-2250 5 (ran out)

35 μL of dH₂O was added to tube 1-1, and a distinct blue color changewas observed quite quickly (>15 minutes). When left out overnight(eppendorf tube uncapped), the color turned to a dark blue/green, asillustrated in FIG. 6.

Two microcentrifuge tubes were left (1-2 and 4-2) that can be tested ata later time to ensure that the pullulan protects the AChE fromoxidation.

IDA in PVA Test (Protection Test)

15 μL of IDA dissolved in methanol was added to a pullulan capsule (castin an eppendorf tube lid), and sealed with a 5 mL (50 mg/mL, 3.5 cmpetri dish) PVA film using a PVA glue solution. Another capsule wasprepared with IDA in a powdered form. The capsules were left todetermine if the PVA will serve as an oxygen barrier to protect the IDAfrom oxidation over a period of time

Creating New Sensor Devices

In order to make a paperless sensor, it is desirable to allow for ˜5minute incubation of the enzyme within the water before the IDA isreleased. In order to do this, it is theorized that the eppendorf tubesthat were previously used can have a thin layer of enzyme cast overtopof a capsule containing IDA in the bottom of the eppendorf tube. Thislayer of enzyme-containing pullulan should be thick enough to notdissolve through the pullulan capsule, and to allow the solution to dry.

The previously formed capsules that were cast in the smallest eppendorftube cap lids, and filled with 5 μL of IDA solution were sealed with a 5mL pullulan film, and the edges of the film cut off. The entire capsulewas then placed into tube 4-1 (containing 10 μL of enzyme).

FIG. 7 demonstrates the visible color change of the solution when 35 μLof dH₂O is added.

Capsule Tests with Pesticide and Control

In order to determine if a similar sensor setup will work (AChE andIDA), and to see if a new standard curve will need to be generated,several tests were performed.

5 μL of AChE was added to 40 μL of 250 mg/mL pullulan solution, and castinto a 0.6 mL Eppendorf microcentrifuge tube. A 2 mm (diam)×4 mm (depth)capsule with an enlarged flange was filled with 5 μL of IDA (capthickness of 5 mL, diam of 5/32″), and added to the Eppendorf tube 5days after casting.

30 μL of dH₂O was added to the tube, to dissolve the capsule and allowfor the reaction to occur.

The resulting color intensity increases over time, and turns a bluecolor, as indicated by FIG. 8. As can be seen, the color intensitychange is lessened after 15 minutes, as the reaction nears completion,and slows down.

To ensure that the observed color change is due to the reaction of IDAwith AChE, samples are tested with an organophosphate pesticide(concentration of 10-3), and two control samples, as demonstrated inFIG. 9. It is evident that there is a color change for the sample tubewith dH₂O, as expected, and at time of 15 minutes for the samplecontaining no enzyme, there is no visible color. However, when the timeis extended to 60 minutes, there is a faint bluish color, that may bedue to possible contamination, or due to an unknown side reaction of theIDA (perhaps with pullulan).

Masses of Pullulan Capsules

The masses of pullulan capsules cast of 250 mg/mL pullulan solution aremeasured, and the average value (with standard deviations) arecalculated in order to determine how homogenous the samples arc. Theresults are indicated below which gives the average mass and standarddeviation for each type of pullulan capsule cast of 250 mg/mL solution.

Type Average ± St. Dev. (mg) Small (holes)  3.80 ± 0.28 Small (no holes) 4.95 ± 0.79 Medium (holes) 11.60 ± 2.44 Medium (no holes) 13.37 ± 3.42

The sources of variation in these capsules are due to variations in thecapsule mold, and possible differences in the volumes added. It issuggested to add a prescribed volume of solution to each of the capsulemolds to ensure the same mass of pullulan results.

Casting IDA into Pullulan

In order to determine if pullulan works to stabilize IDA when the IDA ismixed into the pullulan solution, a solution of 300 mg/mL pullulan wasmixed, and 5 μL of IDA dissolved in methanol was added to each 30 μL ofsample. A thick pullulan solution was used to minimize the amount ofcontact with water the IDA will have, to minimize the hydrolysing (henceinactivation) of the IDA.

Five samples were cast (total volume of 35 μL) into the caps of 0.6 mLEppendorf microcentrifuge tubes and left to dry. The activity of the IDAwill be measured by:

-   -   i. checking for a pink/red color change, which indicates the IDA        is hydrolysed or oxidized    -   ii. testing with AChE to ensure that a blue color change still        results        Pesticide Tests

Three trials were conducted with fresh malathion (from BioninterfacesInstitute), which was diluted with Tris buffer to a concentration of10-2 mol/L. A control sample was also done with 5 μL of AChE and 250mg/mL pullulan to ensure that a color change would occur. The colorchange was initially green (˜2 minutes), then became a blue color whichincreased in intensity over time.

Information about the contents and the results of each Eppendorf tubetest with pesticides is presented below. A control test was performedwith 5 μL of AChE and 30 μL of dH₂O with 5 μL of IDA/methanol, and thecolor change to blue-green was very quick.

AChE Pullulan Volume Incubation No. (μL) (mg/mL) (μL) Time (min) 1 5 25030 15 2 10 125 30 30 3 5 — 40 30

No. Result 1 solution turned cloudy upon addition of the IDA/methanol.No color change at 5 minutes. 2 Slow color change to blue (about 5minutes to be noticeable on camera, 10 to be noticeable by eye), thencolor intensified to near the color of the control sample. 3 Sample wasaccidently further diluted with 5 μL of Tris buffer, and the colorchange was very slow (similar to 2), and eventually resulted in a darkblue color that was several shades lighter than (2). This may be due todilution, or perhaps the fact that the AChE was indeed more inhibited.

Since the AChE acts as a catalyst (not a reagent) in this reaction, itis possible that even if a small fraction of the AChE is not inhibitedby the pesticide (regardless of concentration), the small amount of AChEcould act to degrade a significant proportion of the IDA, but it wouldtake longer. This could explain the anomalous results obtained thus far.On a paper-based device, the immobilized AChE is limited to degrade theIDA that is immediately around it. However, when in solution, the AChEis able to contact with much more of the IDA and degrade it, but itwould take longer.

Reaction Kinetics

In order to know how much of each reagent and enzyme to add to thesolution, it is desirable to know more about how the reaction proceeds.The kinetics (and mechanism) of the hydrolysis of N-methylindoxylacetate is known (M H Sadar, K J Laidler. Transient Kinetics of theAcetylcholinesterase Catalyzed Hydrolysis of N-Methylindoxul Acetate.Can J Biochem. 1974). N-methylindoxyl acetate is structurally quitesimilar to indoxul acetate, with the exception of a single non-reactingmethyl group attached to the nitrogen atom. Due to the structuralsimilarities, then, the kinetics are assumed to be quite similar. Therate law expression of this reaction follows the Michaelis equation as:v=k _(c)*[E]*[A}/(K _(M)+[A])with k_(c)=320 s⁻¹ and K_(M)=2.58×10⁻³ M.

The enzyme and substrate concentrations must be converted to units ofmol/L in order to use the rate law equation. Accordingly, for the enzymeit is known that there is a 500 U/mL concentration in solution, and 513U/mg of the solid powder. Additionally, according to published material(S R Levinson, J C Ellory. The Molecular Form of Acetylcholinesterase asDetermined by Irradiation Inactivation. Biochem J. 127. 1973. 123-125)the approximate molecular weight of a single acetylcholinesterasemolecule is ˜75000 g/mol. Using this information, the concentration ofthe enzyme can be calculated. The concentration of the IDA in thesolution is provided by the prepared IDA solution, then adjusted toaccount for the increased volume in the Eppendorf tubes.

Trials with Different Concentrations

It was attempted to use different concentrations of the enzyme and IDAin order to optimize the color intensity, while ensuring that theintensity is low enough that there is a noticeable decrease in intensitywhen pesticides are applied. Accordingly, several Eppendorf sample tubeswere prepared as outlined below which gives conditions of the differentEppendorf tubes prepared to test the effects of concentration of enzyme.

Sample # [IDA] (mol/L) [AChE] (mol/L) 1-1 4.762 × 10⁻⁴ 3.064 × 10⁻¹⁰ 1-29.302 × 10⁻⁴ 2.993 × 10⁻¹⁰ 2-1 2.174 × 10⁻³ 2.798 × 10⁻¹⁰ 2-2 9.709 ×10⁻⁵ 3.124 × 10⁻¹⁰ 3-1 4.651 × 10⁻⁴ 5.986 × 10⁻¹⁰ 3-2 9.091 × 10⁻⁴ 5.850× 10⁻¹⁰ 4-1 2.128 × 10⁻³ 5.477 × 10⁻¹⁰ 4-2 9.479 × 10⁻⁵ 6.100 × 10⁻¹⁰

The sample sets 1 and 3 were first tested to determine the color changeintensity. It was found that the color change began around the 1 minutemark, and continued to increase in intensity until about 30 minutes. Atthe 15 minute mark, the color intensity was nearly as developed as itwould become overnight. A 15 minute test time is suggested.

The results of sets 1 and 3 were that the maximum color intensity wasobserved for the 1-1 sample. This is unexpected, as it represents thelowest concentrations of both the substrate and the enzyme of all theothers.

Upon analysis of the kinetics information, as pictured in FIG. 10, itwas determined that according to kinetic theory as previously mentioned,the expected order of intensity is 3-2>3-1>1-2>1-1.

However, the results of the color intensity measurements, as performedby ImageJ as previously described⁶, revealed that the expected trend wasnot followed. Rather, as demonstrated, the color intensity of 1-1 wasthe darkest, followed by 3-2, 3-1 and 1-2. It is possible that differentlighting conditions between the trials may have resulted in some randomerror for measuring the average color intensity.

Upon further testing, it was discovered that what appears to be theideal concentration of enzyme and substrate is 1 uL of AChE, 40 uL of100 mg/mL pullulan solution and 1 uL of IDA. The color change is rapid(˜1 minute to begin), and distinctly blue against any background.

Testing IDA Capsules and Pullulan Films

Previously explored was the addition of IDA (solution and powder form)to two PVA capsules. 5 uL of IDA dissolved in methanol was added to one,and IDA in powder form was added to the other. When the samples weretested by the addition of Tris buffer to dissolve the capsule and AChEto measure the activity of IDA, a very distinctive blue color change wasobserved, indicating that the IDA was kept stable over a period of twoweeks (approximately). Additionally, it was noted that the IDA solutionwas rapidly released, and resulted in a color change quite quickly,while the powdered form required a much longer time and required theaddition of more Tris buffer and mixing in order to change color. Overthe weekend, the color intensity was approximately the same however,indicating that it is a kinetic problem, not a molecular stabilityissue.

Also tested was one of the capsules with IDA solution cast into apullulan film (in the lid of a 0.6 mL eppendorf tube). The same distinctblue color change occurred, indicating that the IDA retained itsactivity when protected by the pullulan film. Four more capsules were tobe left, and tested each week to determine if there is a decrease in theactivity of the IDA over a longer time period. For now, it can be statedthat the pullulan protects the IDA from hydrolyzation/oxidation for ˜2weeks reliably.

Comparison of AChE Activity w/ Different Concentrations of Pullulan

To determine the effect the concentration of pullulan has on themaintenance of the AChE enzyme two samples of 2 weeks age were tested(125 and 250 mg/mL). The resulting color change demonstrated that theactivity of the enzyme was not much decreased over a period of twoweeks. More trials will be conducted at 1 week intervals to determine ifthere is a noticeable color intensity difference over time.

Spectrophotometry

As a method of tracking the reaction progress and quantifying the colorchange due to pesticide inhibition a spectrophotometer was used. Thespectrophotometer requires a minimum volume of 700 μL of sample to beplaced into a cuvette, which is then individual read. This is timeconsuming, but provides a quantifiable method of determining the colorintensity (and repeatable method).

For the first experiment, 10 different samples, and one blank sample. 1μL of AChE was cast into the bottom of a 0.6 mL Eppendorf tube, in 200mg/mL pullulan (40 μL volume). The resulting solution then had pesticide(diluted in Tris buffer) added in the volume of 160 μL. For the blanksample, no IDA was added, while for the other 10 samples, 1 μL of IDAwas added (in aqueous form). Pesticide (malathion) was added in variousconcentrations to quantify the inhibitory effect of the pesticide on theenzyme.

FIG. 11 demonstrates the curve that resulted from this experiment,demonstrating that the higher the concentration of pesticide, the lowerthe color intensity that results is. This result is expected, but thereis some variation about the expected trend. A sigmoid curve is fitted tothe data (for the ‘inverse s-shaped curve).

Two wavelengths were tested, the theoretical wavelength of the IDA colorchange (605 nm) and the result of a wider-spectrum scan (725 nm) weretested.

Multiple Well Samples (BI Spectrophotometer)

In order to track the kinetics of the enzyme activity (rather thansimply a color change at one point in time), the 96 well plate withspectrophotometer in Biointerfaces Institute was used. This scannerallows for the absorbance of each well to be measured as a function oftime (scans˜every 9 seconds). A constant amount of AChE (1 μL) was addedto each sample. Each sample contained 40 μL of a 150 mg/mL pullulansolution, and is diluted with 159 μL of Tris buffer (where the pesticidewill eventually be).

Using the plots of the concentration of IDA (substrate) vs. theabsorbance (at 605 nm), the initial rate of the enzyme reaction can becalculated as the k_(m) value of the enzyme (characteristic). The kmvalues can then be plotted onto a curve comparing the k_(m) as afunction of pesticide concentration.

Measurements are taken by Tecan infinite M1000 reader (microplatereader).

Several different methods of solution preparation were used, including:

-   -   i. mixing all the pullulan, Tris and enzyme in an eppendorf        tube, then adding to three separate wells and adding IDA        simultaneously to each well    -   ii. preparing each well independently, and adding the IDA        solution simultaneously to three wells    -   iii. preparing wells independently, and adding the IDA        separately, but keeping track of the timing between each well so        as to adjust the plot.

Different patterns of well that were used in the 96-well plate were alsoexplored, as demonstrated in FIG. 12 below.

Method of Data Analysis

The data returned by the 96-well plate reader (both Tecan Infline M1000and M200 Pro) was analysed by plotting the absorbance of each sample asa function of time. The time scale was adjusted to set the initial timeas the start of reaction, not as the start of measuring (for example,immediately after measuring began, the time reading was 0.00 for everysample. However, the actual time of reaction was up to 3 minutesalready).

The initial slopes of the absorbance curves were then obtained vialinear regression. Two intervals were considered, from 0-500 s and from0-1500 s (reaction time, not readout time). These slopes (units ofabs*s⁻¹) were then plotted as a function of the concentration of IDAadded to each well. A concentration range of 0-5 mM was considered(final concentration).

After a certain time period, the absorbance reaches a plateau, as thereaction reaches completion. The speed of the reaction depends on theamount of enzyme that is present. The sensor papers operated with 4-5 uLof AChE (500 U/mL), but this is far too much for a liquid phasereaction, and even pesticide of concentration 10⁻² M was unable toinhibit the enzyme. Accordingly, an enzyme volume of 1 μL of 250 U/mLwas chosen, and seems to work well. The time of the reaction to reachcompletion (plateau) is about 30 minutes, as demonstrated in FIG. 13.

IDA Auto-Hydrolyzation

The plots that were obtained, however, did not account for theauto-hydrolyzing of the IDA. The wells were diluted with Tris buffer, inwhich IDA will degrade. Accordingly, a blank sample was set up (with noenzyme, but all other reagents similar to the real trials), and theslope of the absorbance vs. time curve was obtained for each IDAconcentration. This slope (related to rate of reaction) was thensubtracted from the slopes of the trials with enzyme, to ensure that thereaction (and color that resulted) was due to the activity of theenzyme, and not the IDA hydrolysis without the enzyme.

For the concentrated IDA solutions that were prepared in methanol (up to200 mM), it was observed that the resulting color after a time periodwas yellow. However, there is a possibility that this color couldinterfere with the spectrophotometer results. FIG. 14 below demonstratesthe slopes of the absorbance vs. time curves for the control samples.

Results of the Well Plate Reader

The results of the well plate reader is a plot of the reaction rates(correlated to abs*s⁻¹) as a function of the concentration of IDAsolution used. FIG. 15 below demonstrates the final plots, as obtainedusing Sigma software for both the 0-500 s and the 0-1500 s case.

Pullulan Interference and Protection

Pullulan in the solution is supposed to protect the enzyme from beinginactivated over time. Accordingly, a trial was conducted in which a5-day old enzyme and pullulan solution was redissolved and added to awell, and tested against a fresh pullulan sample (with the same amountsof IDA and AChE).

Additionally, a sample of Tris buffer with no pullulan is tested, tocompare the effect the addition of pullulan has on the reactionkinetics. Since the pullulan increases the viscosity of the fluid, it isexpected that the result be a slow reaction. FIG. 16 demonstrates theresults.

AchE Stability Over Time

In order to quantify the protection that pullulan provides to AChE overtime, it was attempted to create a set of samples (40) which will betested over time. Each sample (cast into a 600 μL eppendorf tube)contains:

i. 40 μL of 200 mg/mL pullulan solution

ii. μL of AChE (250 U/L)

Each sample tube was allowed to dry, then was sealed and placed into aplastic bag and stored at ambient conditions (˜20° C.). At selected timepoints, the activity of the solution was tested by measuring absorbanceover time, and finding the associated reaction rate (corresponding tothe activity of the enzyme). To measure the activity, three (3) sampleswere taken (to establish triplicate repeats), and 195 μL of Tris buffer(100 mM, pH=8.0) was added to each Eppendorf tube and allowed todissolve the pullulan/IDA film. The resulting solution was then added toa 96 well plate, and absorbance was measured continuously at 605 nmafter the addition of 5 μL of 80 mM IDA. The results of these trials aredemonstrated in FIG. 17.

Different Pullulan Concentrations

Trials were also conducted with different pullulan concentrations todetermine which pullulan concentration results in the best protection ofthe enzyme. 40 μL of each pullulan solution was added with 1 μL of AChE(250 U/L) and cast into the lids of a 600 μL Eppendorf tube.

The resulting film/capsule/pill was then used by dissolving it into 195μL of Tris buffer (100 mM, pH=8.0), and pipetted into a 96 well plate,where 5 μL of IDA (80 mM) was added to the well. The absorbance wasmeasured over time to obtain a resulting reaction rate. The finalresults are shown in FIG. 18.

Samples were left for 4 days in the refrigerator to be casted, and thentested. Error bars are presented as one standard deviation based ontriplicate repeats. 40 μL of pullulan solution (various concentrations)was used, and 5 μL of 80 mM IDA was added to each sample. The resultingdata does not show any statistically significant trends, due toexorbitantly high error bars (due to so many source of error andvariation in experimental setup). The data does suggest an increase inthe rate of reaction which peaks at a pullulan concentration of 100mg/mL. Therefore at lower pullulan concentrations, the film formed istoo thin to protect meaningful amounts of enzyme, while at higherconcentrations, the increased solution viscosity hinders diffusion andmixing properties, as well as extends the drying time of the capsule(increasing risk of enzyme degradation by oxygen/heat/etc).

Ellman Assay in Solution & on Paper

While indoxyl acetate (IDA) does provide a good substrate to measure theactivity of AChE, the Ellman assay (using ATCh and DTNB as substrates)is much more widely used in enzyme research. Accordingly, the necessaryreagents were obtained, and preliminary testing began.

When tested in solution, with the following amounts of reagents wereused:

i. 15 uL ATCh (300 uM), 1 uL AChE (10 or 250 U/L)

ii. 50 uL of 500 uM DTNB

iii. 34 uL Tris

It was noted that the color change (yellow) in solution remained forsome time before fading. However, when a paper strip was dipped into thesolution and allowed to dry, the color faded overnight. This may bepartially due to the reaction products being unstable on a papersubstrate, as well as difficulty identifying a light yellow color changeon a white background.

Acetylthiocholine (ATCh) is an unstable molecule, and will hopefully bestabilized on paper. However, after several tests were performed, it wasnoted that after being left in the refrigerator for several days, andleft in a fume hood with no protection against hydrolyzation, the ATChwas still stable when tested. The most likely reason for the continuedactivity of the ATCh is due to the Tris buffer it was dissolved in beingevaporated, and the ATCh in a solid (or powdered) form is stable.Accordingly, it should be reliable to store the ATCh as a powder form,preferably inside of a soluble (pullulan) capsule.

Example 3: Further AChE/IDA Bioassays

Materials

Acetylcholinesterase (AChE, from Electrophorus electricus, EC 3.1.1.7),and indoxyl acetate (IDA) were obtained from Sigma-Aldrich. Pullulan (MW˜200000) was purchased from Polysciences, Inc and malathion was obtainedfrom Fluka. Human serum albumin (HSA; fatty acid and globulin free,≥99%) was obtained from Sigma-Aldrich (Oakville, ON). Quartz microscopeslides were purchased from Chemglass (Vineland, N.J.) and cut toapproximate dimensions of 8×32 mm. Water was purified with a Milli-QSynthesis A10 water purification system. Buffer salt (Tris 100 mM) andpullulan solutions were filtered using a Pall® syringe filter with 5 μmmembrane in order to remove any dust particulate.

Experimental Procedures

IDA Tablets, Creation and Activity Test.

To test the ability of pullulan to retain IDA activity, 10 μL of 40 mMIDA and 40 μL of 120 g/L pullulan in water were mixed and casted in apolypropylene mold with wells with a size of 3 mm in diameter×3 mm indepth. The solution was air-dried overnight at 21° C. and 48% RH; tabletformation was considered not fully completed if the tablet could not beremoved from the bottom of the well. The resulting tablets were kept atroom temperature for different lengths of time before being tested. Totest IDA activity, 199 μL of Tris-HCl (100 mM, pH 8) was used todissolve the pill and 1 μL of fresh 250 U/mL AChE was added. Thesolution was then transferred to a 96-well plate and the absorbance ofthe developing blue color was measured at A₆₀₅ on a TECAN Infinite M200Pro microtiter plate reader.

AChE Tablets, Creation and Activity Test.

To test the ability of pullulan to retain AChE activity, 1 μL of 250U/mL AChE and 40 μL of 120 g/L pullulan were mixed and casted intotablets. The solution was air-dried overnight at 21° C. and 48% RH;tablet formation was considered not fully completed if the tablet couldnot be removed from the bottom of the well; and the resulting tabletswere kept at room temperature for different lengths of time before beingtested. To test AChE activity, 195 μL of Tris-HCl (100 mM, pH 8) wasused to dissolve the tablet and 5 μL of fresh 80 mM IDA was added. Thesolution was then transferred to a 96-well plate and the absorbance wasmeasured at A₆₀₅ on TECAN Infinite M200 Pro microtiter plate reader.

Malathion Detection Test.

200 μL of malathion solution was added into the Eppendorf tube followedby the addition of the pullulan-AChE tablet. After 5 minutes ofincubation, the pullulan-IDA tablet was added into the Eppendorf tube.The mixture was incubated for 10˜15 minutes for development of the bluecolor; the concentration of malathion was calculated based on the colorintensity. Images were obtained using a Galaxy Nexus cellphone cameraoperated in automatic mode with no flash. The Images were analyzed usingImageJ software by methods described elsewhere⁶. The concentration rangeof malathion was 0.01 to 1E-10 M.

Preparation of Pullulan-HSA Solutions and Films.

Human serum albumin (60 μM final concentration) was dissolved intoeither 100 mM Tris-HCl (pH 7.5) or 100 mM Tris-HCl containing 10%pullulan. These pullulan solutions, with or without HSA, were carefullypipetted onto the quartz slides (500 μL/slide) and allowed to dryovernight at 21° C. and 48% RH in order to produce the film samples.

Determination of the Water Content in Pullulan Films.

The water content of films was done by gravimetric analysys with dryingat 130° C. until constant weight. The water content in the films wasfound to be 0%.

Fluorescence Intensity Measurements.

Fluorescence measurements were acquired using a Cary Eclipsefluorescence spectrophotometer. Solution samples were measured in quartzcuvettes and continuously stirred throughout the experiments. Filmsamples were suspended in quartz cuvettes at a 45° angle to theexcitation light using specialized holders which reflected excitationlight away from the detector and collected emission through the slideand into the monochromator/PMT.

For fluorescence emission spectra, samples were excited at 295 nm (toensure that the light was absorbed almost entirely by the lonetryptophanyl residue) and emission was collected at 310-450 nm in 1 nmincrements, using a 5-nm bandpass for both excitation and emission pathsand an integration time of 0.1 s. Spectra from both solution andfilm-based samples were corrected for light scattering by blanksubtraction of signals originating from buffer or pullulan/quartzmaterials, respectively, without HSA. All the spectra were alsocorrected for deviations in emission monochromator throughput and PMTresponse and smoothed by the Savitzky-Golay method, using a factor of 5and an interpolated factor of 5.

For thermal denaturation studies, the temperature was raised in ˜5° C.increments from 20° C. to 90° C. and allowed to equilibrate at eachtemperature for at least 5 min. The temperature in the cuvette wasmeasured directly with a thermistor probe. Intensity-based unfoldingcurves are reported as integrated scan intensities, which are normalizedto the integrated intensity at the beginning of the experiment (20° C.as 100%) for each sample. Emission scans are measured in relativefluorescence units, RFU, and all values are reported as the average ofthree separate samples.

FIG. 19 shows a graph of the reaction rate (abs/s) of AChE and IDA as afunction of the IDA concentration from 0 to 4 mM. The reaction rate ofAChE tablet with different concentration of IDA was monitored toestablish an optimal IDA concentration for pesticide detectionexperiment. From the data, the concentration of IDA for the pesticidewas chosen to be 2 mM (which is significantly larger than KM). The errorbars represent the standard deviations based on triplicate repeats.

FIG. 20 shows the fluorescence intensity-based thermal unfolding curvesfor HSA-buffer solution, in HSA-pullulan solution and HSA-pullulan film.Changes in the intrinsic fluorescence from tryptophan (Trp) residueswithin proteins can be used to provide information on proteinconformational stability and unfolding⁷. Therefore, steady-statefluorescence spectra were measured at various temperatures forHSA-buffer solution, HSA-pullulan solution and HSA-pullulan film (HSAwas chosen for this study because it contains a single Trp allowing forunambiguous investigations. The data shows that the intensity offluorescence for HSA-buffer and HSA-pullulan solution decreased by morethan 90% when the temperature was raised from 20° C. to 90° C., whilethat of HSA-pullulan film only decreased by ˜70%. The unfoldingtemperature (wherein the Trp intensity is reduced by 50%) wassignificantly higher for the pullulan-HSA film at ˜80° C., versus ˜60°C. for both HSA-buffer and HSA-pullulan solutions. This studydemonstrates that pullulan film significantly enhances the thermalstability by preventing substantial unfolding as a result of molecularconfinement in the rigid matrix. This stabilizing effect is onlyobserved in the pullulan film and not in the pullulan solution.

Discussion

Individual AChE-pullulan tablets and IDA-pullulan tablets were producedby using a process that involves 1) the mixing of a pullulan solutionwith either an AChE or IDA solution, 2) the casting of each mixture intoa polypropylene mold with small wells (3 mm in diameter_3 mm in depth),and 3) air-drying. Note that defined concentrations of AChE and IDA werechosen to achieve a maximum rate of color formation (see FIG. 19). Toconduct the assay, an AChE tablet was added to the sample to allowpreincubation with the pesticide followed by the addition of an IDAtablet. If malathion is present, the sample remains colorless or turnsfaint blue (dependent on the concentration of malathion, as discussedbelow). In the absence of malathion, IDA is fully hydrolyzed by AChE andthe test sample turns deep blue.

The tablet system can not only be used achieve qualitative colorimetricdetection of malathion by eye, it can also provide quantitative analysisof the pesticide concentration in a test sample when using a smartphoneand image-processing software (such as ImageJ⁶). FIG. 21 shows a plot ofthe dose-dependent inhibition of AChE by malathion, with data obtainedusing a smartphone. This simple method can be used to detect malathionat levels as low as 64 nm (S/N=3).

The long-term stability of both the AChE and IDA tablets was tested. Asshown in FIG. 22, AChE stored in solution at room temperature becamecompletely inactive within 3 days. Similarly, IDA in solution at roomtemperature lost 70% of its activity within one day and becomecompletely inactive within a week. In sharp contrast, both AChE and IDAin tablet form remained fully active for at least 2 months when storedat room temperature. In the case of IDA, the loss in performance wasrelated to oxidation._([16]) Data suggests that pullulan acts as astrong barrier to oxygen, an effect that is consistent with previousfindings._([7, 9]) In theory, an antioxidant could be used to preventthe oxidation of IDA during storage at room temperature. However, theantioxidant would also inhibit the formation of indigo during the assay.

The loss of AChE activity, however, is attributed to thermaldenaturation. To further examine the role of pullulan in stabilizingAChE, activity of AChE was monitored as a function of temperature. Forthis experiment, native (unencapsulated) AChE and the correspondingpullulan tablet were treated at a given temperature for 30 min, followedby activity assessment at room temperature. FIG. 23 shows that the freeAChE became completely inactive following a 30-minute heat treatment at50° C. or above. In stark contrast, AChE tablets retained ca. 90% oftheir initial activity even after a 30-minute incubation at 90° C.Significant thermal stabilization was also observed for human serumalbumin (FIG. 20) where the unfolding temperature of the protein, asdetermined by tryptophan emission intensity, increased by 20° C., thusdemonstrating that the stabilizing effects of pullulan are generic.

Example 4: Highly Stable ‘All-In-One’ Bioluminescent Pill for SensitiveATP Detection

Materials.

Luciferase, Luciferin, Co-enzyme A (CoA), Adenosine triphosphate (ATP),Tricine, Magnesium Carbonate (MgCO₃), Magnesium Sulfate (MgSO₄),DL-Dithiothreitol (DTT), Ethylenediaminetetraacetic acid (EDTA), andDextran (Mw ˜148000) were purchased from Sigma-Aldrich. Polyethyleneglycol (PEG, Mw ˜6000) was purchased from Fluka. Pullulan (Mw ˜200′000Da) was purchased from Polysciences.

Preparation of ‘All-In-One’ Pullulan Pill.

All reagents for the luciferase assay except for adenosine triphosphate(ATP) were casted in a single pullulan pill. For the pullulan pillsaqueous solutions of 10 mM Luciferin, 100 mM Luciferase, 27 mM CoenzymeA (CoA), 170 mM Dithiothreitol (DTT), 10 mM Ethylenediaminetetraaceticacid (EDTA), 107 mM MgCO₃, and 267 mM MgSO₄ were prepared. 200 μL ofeach solution was added to 8 mL of 12 w/v % Pullulan solution. Lastly,for each pill 47 μL of the final solution was pipetted onto a PET filmand dried in a glove box under nitrogen. The casted pills were thenstored at room temperature.

Buffer Preparation.

A buffer solution containing ATP was prepared to test the activity ofthe luciferase pills. For the buffer solution, 16 mL of water was addedto 2 mL of 2.5 mM ATP and 2 mL of 200 mM Tricine and the pH was adjustedto pH 7.8.

Stability at Room Storage Condition.

Once the pills were completely dried, they were collected in darkbottles. The pills were stored at room temperature and the activity ofthe pills was tested over time. On the day the pills were prepared, theactivity of the fresh pullulan and reagent solution was tested. 47 μL ofthe solution and 100 μL of the buffer were added to a 96 well-plate andthe luminescence was measured using the TECAN M1000. On subsequent days,the activity of the pills was measured by placing a single pill into awell and 100 μL of ATP buffer. Each test was performed with threerepeats.

ATP Detection Assay.

The sensitivity of the luciferase assay was also investigated. Todetermine the detection limit of the assay, different ATP buffers wereprepared with concentration ranging from 0 μM to 1000 μM and tested itwith luminescent pills.

Thermal Stability.

Reagents in solution and pullulan pills were incubated at eachtemperature set point for 30 minutes in a hot plate. After that, theywere allowed to cool until room temperature was reached. Then theluminescence reading was taken using 100 uL of 250 uM ATP (in tricinebuffer)

Stability in Dextran/PEG.

The effect of other polymers and polysaccharides on luciferase activitywas investigated. Dextran and PEG pills were prepared using the sameprocedure as the pullulan pills. In place of pullulan solution, 12 w/v %Dextran and 12 w/v % PEG solutions were used to create dextran and PEGpills respectively.

For comparison, the same study was repeated with dextran andpolyethyleneglycol as additives. (PEG and Dextran cannot be used asstabilizer as they do not preserve the luciferase, data not shown).

Cell Assay Using Luminescent Pill:

E. coli DH5α cell cultures were started from a glycerol stock and grownin Müller Hinton (MH) media at 37° C., 250 rpm, for 18 hours. A 1:50dilution of the overnight culture in MH broth was created and grownuntil log phase (OD₆₀₀=0.3). At this point, a 10 μL of the culture wasserially diluted by 10⁵ in sterile PBS and plated, in triplicate, on LBagar plates for enumeration. Cell lysis was performed right before theluminescence testing by adding B-Per in the volume ratio of 1:10 (B-Per:Cell culture) and was incubated for 15 minutes. 100 uL of the lysedcells was added into the well containing luminescent pill.

LC-MS Analysis:

LC Method:

Phenomenex C18 column 100×3 mm dimensions with 3 um particle size wasused as stationary phase. 10 mM Ammonium acetate was used as mobilephase with acetonitrile. 14 minutes gradient with acetonitrile goes upto80% in 10 minutes. Chromatogram was monitored in 254 nm wavelength withUV detector during method development.

Sample preparation was done by dissolving luciferin pill with 50 uL ofwater followed by 500 uL of methanol. The precipitated pullulan wasfiltered using 0.2 um syringe filter.

MS Condition:

Ionization:

ESI negative mode, Capillary Voltage: 400 V with Quadruple analyzer.

Discussion

In this work, pullulan, a polysaccharide comprising maltotriose unitswas used to form ‘all-in-one’ luminescent pill for ATP detection. Allthe components such as luciferase, luciferin, MgSO₄, MgCO₃,dithiothreitol, ethylenediaminetetraacetic acid (EDTA), CoA enzyme andtricine buffer, for the luminescence detection of ATP were taken inpullulan (12% w/v final concentration) and encapsulated to form a stable‘ready-to-use’ pill. Encapsulation with pullulan not only protects thecomponents because of its oxygen barrier property and polymer crowdingbut also forms a stable, non-hygroscopic and size/shape-tunable pillsafter drying. When the luminescent pill was treated with ATP insolution, it readily dissolves and releases all the components forluminescence detection (FIG. 24).

In the present work, the pill-making process involves simple mixing thecomponents and drying in an inert atmosphere to give the luminescentpills. Unlike other polymers used previously for this purpose, pullulanhas an additional oxygen barrier effect to it. To the best of theinventor's knowledge, this is the first report in which all thecomponents required for luciferase/luciferin assay were stabilizedtogether as a single ‘drop and detect’ pill for the ATP detection. Theconcentration of pullulan for casting the pills was optimized based ontwo factors viz., retention of glow kinetics and minimum drying time.The glow kinetics was retained between 0.001 mg/mL and 10 mg/mL ofpullulan concentration and started decreasing when it the concentrationwas increased to 100 mg/mL in solution. Also, low concentration. such as0.001 mg/mL, will have a longer drying time which affects the stabilityof the encapsulated enzyme. The stability of the pills at roomtemperature was evaluated by storing the pills at bench top in darkcontainers. FIG. 25A shows the glow kinetics (luminescent units versustime) of a luminescent pill with 250 uM ATP concentration on a 96 wellplate reader (Tecan M1000). FIG. 25B shows the stability of pills incomparison with the same components stored in buffer. This shows thatthe enzyme loses its activity in buffer solution in few hours. On theother hand, the enzyme and other components in pullulan pills were foundto be stable for 3 weeks at bench top. The most unstable components inthe pill are enzyme (luciferase) and luciferin. The stability of enzymealone was also tested in pullulan pills and was found to be active evenafter four months when stored at room temperature, which led to thestudy of the luciferin-pullulan pill by a suitable technique. which inthis case is LC-MS.

Luciferin, as a substrate for this oxidative-decarboxylation reaction isalso unstable at room temperature and prone to auto-oxidation andphoto-degradation. One of the potential inhibitors of luciferase is thedehydroluciferin which forms when luciferin is exposed to air. Toconfirm the exact degradant responsible for the decrease in activity,luciferin alone was casted as pills with pullulan. Four weeks oldluciferin pill was taken for LC-MS analysis. When luciferin wasextracted with methanol by dissolving the pills with a small amount ofwater and then with methanol. Pullulan precipitated because of its poorsolubility in methanol. Then the suspension was filtered using 0.2 uMsyringe filters. The filtrate was expected to have luciferin and itsdegradant because of its high solubility in methanol. The challengingpart was the impurities associated with pullulan were also present inthe filtrate as commercial pullulan is not 100%, resulting in too manypeaks. Hence, it was decided to use extracted ion chromatogram (EIC) forthe key impurities by its m/z ratios. In ESI-MS, pure luciferin inmethanol shows up as a single peak at 4 minutes which correlates to twomass peaks, luciferin 279 Da and its decarboxylated product at 235 Da(fragment under ESI condition) in negative mode. Fresh and four weeksold luciferin pills were first analyzed for the above peaks whichconfirmed its presence. Then, they were analyzed for m/z 277(dehydroluciferin) and 233 (decarboxylated form). The extracted ionchromatogram (EIC) clearly shows the 233 peak in the four weeks old pillat retention time 5.8 min which was absent in fresh. This confirms thatluciferin is converted into dehydroluciferin and/or its decarboxylatedform after three weeks which could be a strong reason for the decreasein stability of the luminescent pills after 3 weeks. However, by dryingand packing the pills in inert conditions, such as inside dry-box, thisproblem will be overcome and shelf-life increased.

As these pills encapsulate all the required components, they are highlyadvantageous as they allow the circumvention of laborious preparationseach time of us. It was observed that polymers such as dextran andpolyethylene glycol were not able to retain the activity that pullulanprovided when they were prepared and tested similarly, which showspullulan has unique stabilizing effect when compared to other polyols.

The thermal stability of luciferase is known to be very low. Theluminescent ‘all-in-one’ pills were also tested for thermal stability byincubating the pills at temperatures up to 70° C. The activity of thepills remains the same up to 70° C. while in solution it decreases evenat 30° C. and lost all its activity at 50° C. This clearly shows thatthe pullulan protection for luciferase also ensures the retention ofactivity at elevated temperatures. The ability of luminescent pills forATP detection was shown in FIG. 26. A series of concentrations of ATP intricine buffer were added to the pills and the luminescence intensitywas measured in a plate reader. The relative luminescent units (RLU)were plotted for the range of concentration. The limit of detection ofATP concentration was calculated to be 16 pM at 3° above backgroundwhich correspond to 1.6 fmole ATP in 100 uL which was added to the pill.This value is relatively better in comparison with the recently reportedmethods⁹. Also, the ability of this pill to detect the lysed E. Colicells was tested and compared with the intact cells (FIG. 27). Thedetection limit was found to be 1800 CFU for the lysed cells which iscomparable to other methods which do not involve amplification orenrichment steps¹⁰. None of the previously reported methods were asstraight-forward and user-friendly as that reported here.

Example 5: Automating Multi-Step Spot Tests Using Integrated Layering ofReagents

Materials

Acetaldehyde, Sodium nitroprusside, Sodium carbonate, TSB (Tryptic SoyBroth), poly-arginine (MW>70 kDa), CPRG (chlorophenolRed-β-D-galactopyranoside, and Allura Red were received fromSigma-Aldrich. Pullulan (P120, molecular mass of 200 kDa) was obtainedfrom Hayashibara Co, Ltd, Okayama, Japan. Hydrophobic spray (Heavy-DutyWater Proofer, SOFSOLE, available at sport accessories stores). PETfilms (Polyester 50 Film, 0.004″ thick, clear, Part #8567k44) werepurchased from McMaster-CARR (Hamilton, ON, Canada).

pH Adapter

A pH adapter was constructed to demonstrate the sequential release ofpullulan films on a paper-based device. The pullulan films were preparedby casting pullulan solution on a PET sheet. Strips of 0.5 cm×2 cm tapewere placed onto the PET sheet and the PET sheet was treated with ahydrophobic spray. The strips of tape were then removed, thus leavinghydrophilic areas on the PET sheet for casting the pullulan films. Inorder to create pullulan films with different pH, several 12% pullulansolutions were prepared with pH ranging from 2-12. HCl and NaOH wereused to adjust the solution to the desired pH. 150 μL of the pullulansolution was then added to the hydrophilic area using a pipette. Thesolution was then air dried to form a film, (see FIG. 28).

The pullulan films were then layered together with films at differentpH. Given the self-sealing property of pullulan, 1 μL of 20% pullulansolution was placed on each corner of the film in order to secure thepullulan films onto each other. In the same way, the stack of pullulanfilms was placed onto a pH indicator paper.

To run the test, the solution was passed through the paper, and thechange in pH was observed by the colorimetric changes of the pHindicator paper. The time of the pH change can be delayed or regulatedby simply adding neutral pullulan films.

Sensor for the Detection of E. Coli.

The sensor comprises two parts: a B-PER pullulan disc and a CPRG paper.

The CPRG Paper.

2 wt % poly-arginine (MW>70 kDa) and 9 mM CPRG (chlorophenolRed-β-D-galactopyranoside) dissolved in water were sequentially printedonto the Whatman #1 filter paper using Canon thermal inkjet printer withthe “high print quality” setting.

The B-PER Pullulan Disc.

B-PER lyses any bacterial cells in the sample solution and in thepresence of β-galactosidase, CPRG will be hydrolyzed to produce ared-colored product. The B-PER buffer solution was prepared by adding 2μL of lysozyme and 2 μL of DNase to 1 mL of B-PER reagent. Each B-PERpullulan disc was then created by casting 40 μL of 50:50 (V/V) mixtureof 10% pullulan/B-PER buffer solution on surface of flexible PET sheetsand air dried. The CPRG paper, which was prepared by printing CPRG ontoWhatman paper, was cut to match the size of the B-PER disc. The B-PERdisc was then attached to the CPRG paper using 1 μL of 20% pullulan(works as a glue).

To run the test, the sensor can be placed into a container such as a96-well plate or a 1.5 mL Eppendorf tube cap. Once inside the container,200 μL of sample solution was added onto the sensor (assay kit) andincubated for 40 minutes. The sensor turns a red/purple color in thepresence of E. Coli and a light yellow color in the absence of E. Coli.The air dried test papers were then imaged using a handheld scanner(Flip Pal 100C Mobile scanner). The color intensity of the sensing zonewas quantified using ImageJ software as described elsewhere⁶.

E. coli Culture.

E. coli (ATCC 25922) was cultured overnight in TSB (Tryptic Soy Broth)media for about 20 hrs at 37° C. with a shaking rate of 200 rpm. (The 5ml culture was started with one colony picked from an agar plate. Noantibiotic.)

Sensor for the Detection of Secondary Amine

Acetaldehyde Pullulan Film.

0.5 g of pullulan was dissolved in 5 mL of water. 1 mL of acetaldehydewas added and mixed using a vortex. It was casted as thin films on thebench top by pipetting out 10 uL drops on PET sheets.

Sodium Nitroprusside-Sodiumcarbonate Pullulan Film.

0.1 g each of sodium nitroprusside and sodium carbonate were dissolvedin 5 mL water. 0.25 g of pullulan was added and mixed using a vortex. 1mL of acetaldehyde was added and mixed using a vortex. It was casted asthin films on bench top by pipetting out 10 uL drops on PET sheets.

For both lateral flow paper sensor and z-direction flow sensors, thefilms were stacked in such a way that the analyte will react withacetaldehyde first followed by SNP and sodium carbonate.

Pullulan Kinetics—Absorbance Measurement

The pullulan films were made by mixing 1 mL of 20 g/L dye (Allura Red orTartrazine) solution with 12 mL of pullulan solution. 2 mL, 4 mL, and 6mL of the mixed solutions were then cast onto three different petridishes to create films with varying thicknesses. The solution in thepetri dishes was air dried. Once dried, the films were removed from thedishes and weighed. Films were then hole-punched to produce 1 cm×0.5 cmcut-outs. These cut-outs were weighed and the mass of dye in the cutouts was calculated. A single pullulan film cut-out was added to a 10 mLvial of DI water. From this, 150 μL samples were taken every 15 secondsand placed in Eppendorf tubes. 100 μL of the sample was then added to a96-well dish. The first sample was taken at time zero, before the filmwas placed into water. These samples were used to determine theabsorbance of the solution at each time to characterize the dye releasekinetics. FIG. 29 shows the total mass of Allura Red released from thepullulan film as a function of time for different film thicknesses

Furthermore, as depicted in FIG. 30, a similar kinetic study was donefor CMC (Sodium carboxy methyl cellulose, MW 250,000), MC (Methylcellulose), PVA (Polyvinyl amine, MW 125,000, 87-89% hydrolysis), andHEC (Hydroxylethyl Cellulose). This demonstrates that, depending on theapplication, different polymers may be combined with pullulan films toprolong the releasing process.

Discussion

Simon's Test

The Simon's test is one of the most widely used methods to detectcontrolled substances such as methamphetamine, and other amphetamines.The test uses acetaldehyde to react with secondary amines to form aninitial enamine product, followed by sodium nitroprusside and sodiumcarbonate to produce an immonium ion, which subsequently hydrolyzes togive the blue-colored Simon-Awe complex. This test is typicallyperformed through the addition of individual reagents to the testsubstance in the sequence described above, leading to a color change inthe case of a positive sample.

The selection of pullulan concentration for each reagent-pill was basedon the right balance between the stabilization effect and the release ofthe reagents. Higher concentrations (>12%) lead to the formation ofthicker pills which will take relatively longer time to dissolve andrelease the reagents. Also, the stabilization of reagents, especiallyacetaldehyde at low concentration (<5%) of pullulan was not optimal,therefore, acetaldehyde pills were prepared from 10% pullulan solutionand the SNP/sodium carbonate pills were prepared from 5% pullulansolution. To produce the multi-step assay, two issues were investigated.First, the initial reaction involves a volatile reagent, acetaldehyde,which normally would not be amenable to immobilization onto a sensor.Second, pre-mixing of the reactants was to be avoided before addition oftest substance. and sufficient time for each step to occur prior to theintroduction of the secondary reagents was needed as an incompleteinitial reactions would lead to a decreased signal. The above concernswere addressed by trapping the reagents in a solid pill using pullulan,which stabilizes the reagents and also keeps them unreacted with eachother, when those pills are stacked.

FIG. 28 shows the reaction sequence for the Simon's test. (FIG. 28a ),along with results obtained using a conventional lateral flow assay(FIG. 31b ) and the z-directional multi-step spot test (FIG. 31c ). Theassays were performed using diethylamine (DEA) as a surrogate formethamphetamine to avoid the need to handle controlled substances. Forthe lateral flow test, acetaldehyde and sodium nitroprusside (SNP) wereentrapped in separate pullulan films that were placed sequentially alonga paper channel in the x-direction (FIG. 31b ). When the analytesolution (DEA in water) was wicked through this channel, the reactionsteps occurred in the expected sequence and produced a blue-coloredproduct. However, the product spread over a significant area of thepaper, and thus the color intensity was decreased. When the same assaywas performed using the z-directional spot test, a far more intensecolor was formed that was retained in a small area owing to the use of ahydrophobic barrier to contain the aqueous sample. The incorporation ofthe Simons test into this format allows for a simplified, reliable andrepeatable method of testing for methamphetamines, requiring minimalresources and personnel training. Further, the required reaction andcolor development time of ˜10 seconds for the spot test format whichmeans that this format is comparable to the liquid phase tests typicallycarried out for Simon's test.

As shown in FIG. 32, the sensitivity and detection limit for thez-directional test is also superior to that of the lateral flow testwhen each sensor is challenged with a series of DEA solutions of varyingconcentration. For the z-directional assay the detection range isbetween 100 ug/mL to 50 mg/mL, while for the lateral flow test the rangeis 1 mg/mL to 10 mg/mL. Another benefit of the z-directional assay wasthe ability to use extremely small volumes of test samples (20 uL) toperform the assay, instead of volumes of ca. 200 uL for the lateral flowtest, which minimizes sample dilution and improves detection limits.Finally, the z-direction test required only 10 seconds to perform, whilethe lateral flow test required ˜11 minutes, improving assay time.

E. Coli Assay

As a second example that highlights the potential for on-sensor samplepreparation, the capability of the z-directional paper-based sensor toinitially lyse bacterial cells to release an internal enzyme(β-galactosidase) followed by colorimetric detection of the releasedenzyme using the chromogenic substrate chlorophenol redβ-galactopyranoside, was examined by adapting a previously reportedmulti-step assay^([5]) to the automated sequential assay format. Thesensor comprises B-PER, lysozyme and DNase cast into a pullulan filmthat is positioned on top of CPRG treated paper. The resultantpaper-based sensor can detect the presence of E-Coli by simply applyingthe sample solution onto the sensor in a one-step process.

FIG. 33 shows the formatting of the E. coli detection assay in thez-direction that allows sample preparation (cell lysis) and reportingwithout user intervention. FIG. 33A shows top view of the basiccomponents of the z-directional test, which comprises a pullulan filmloaded with a detergent (B-PER), lysozyme and DNase I for cell lysis andenzyme extraction and a paper disk containing CPRG (the substrate forβ-galactosidase) and a poly-arginine underlayer to enhance thecolorimetric signal. FIG. 30B shows the side-view of the sensor, withthe composition of the films and reagents. FIG. 33C shows colorintensity results obtained from the assay as a function of bacterialcounts, while FIG. 33D shows alternative assay formats for E. colidetection using paper disks, Eppendorf tube caps or 96-well plates,demonstrating the versatility of the z-directional assay approach.

As shown in FIG. 33C, the fecal coliform test can be run simply byintroducing the water sample to the assay kit followed by 40 minincubation. The sensor will turn a red/purple color in the presence ofE. coli and a light yellow color in the absence of E. coli. To achievequantitative colorimetric detection of E. coli smartphone camera ormobile scanner and image-processing software (such as ImageJ⁶) can beused. FIG. 33C shows a plot of the color intensity versus concentrationof E. coli. This simple, equipment free method can be used to detect E.coli at levels as low as 5×10⁵ cfu/ml.

Example 6: PCR with TAQ DNA Polymerase from Pullulan Pill

Materials.

Pullulan (MW ˜200000) was purchased from Polysciences, Inc. Water waspurified with a Milli-Q Synthesis A10 water purification system. Buffersalt (Tris 100 mM) and pullulan solutions were filtered using a Pall®syringe filter with 5 μm membrane in order to remove any dustparticulate. DNA oligonucleotides were purchased from Integrated DNATechnologies (IDT) and purified by 10% denaturing polyacrylamide gelelectrophoresis (dPAGE) before use. Agarose was obtained from Bioshop(Burlington, Canada). The fluorescent images of gels were obtained usingTyphoon 9200 variable mode imager (GE healthcare) and analyzed usingImageQuant software (Molecular Dynamics). Taq DNA polymerase (TaqDP) wasacquired from Biotools.

Forward Primer: 5′-CAGGT CCATC GAGTG GTAGG A Reverse primer:5′-GTACG TTCAG GAGCA GTGCG A Template:5′-CAGGT CCATC GAGTG GTAGG AGGAG GTATT TAGTG CCAAGCCATC TCAAA CGACGTCTGA GTCGC ACTGC TCCTG AACGT AC

this is for PCR.

PCR.

The DNA was amplified by PCR. Each reaction mixture (50 μL) containedTaq polymerase (1.5 U), forward and reverse primer (1 μM each), dNTPs(0.2 mM each of dATP, dCTP, dGTP and dTTP), and template (10 nM). Inorder to produce the Taq-pullulan tablet samples, the Taq polymerase wasdissolved into 20 μL of Milli-Q water containing 10% pullulan andallowed to air-dry overnight at 21° C. and 48% RH. DNA amplification wasperformed by using the following conditions: 30 s at 94° C., 45 s at 50°C., 40 s at 72° C., 18 cycles. PCR product was analyzed by 3% agarosegel electrophoresis.

FIG. 34 shows agarose gel results of PCR products in the presence ofdifferent pullulan concentrations. The effect of pullulan concentrationon the activity of TaqDP was studied in this experiment. 20 μL ofpullulan solution (50, 100, 150, 200 mg/mL) and 1 μL of TaqDP werecasted into Eppendorf tubes. The activity of TaqDP was tested after oneweek. The results show that higher concentrations of pullulan up to˜10-12% provide better protection of TaqDP. Inefficient protection inhigher concentrations of pullulan (≥20%) is due to high solutionviscosity which causes greater error in the experiments and loosingsamples during pipetting.

While the present application has been described with reference toexamples, it is to be understood that the scope of the claims should notbe limited by the embodiments set forth in the examples, but should begiven the broadest interpretation consistent with the description as awhole.

All publications, patents and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety. Where a term in the present application is found to bedefined differently in a document incorporated herein by reference, thedefinition provided herein is to serve as the definition for the term.

FULL CITATIONS FOR DOCUMENTS REFERENCED IN THE APPLICATION

-   ¹ a) M. Shibata, M. Asahina, N. Teramoto, R. Yosomiya, Polymer 2001,    42, 59-64; b) R. S. Singh, G. K. Saini, J. F. Kennedy, Carbohydr.    Polym. 2008, 73, 515-531; c) A. Imeson, Food Stabilisers, Thickeners    and Gelling Agents, Wiley, Hoboken, 2011, pp. 267-274.-   ² S. Farris, L. Introzzi, J. M. Fuentes-Alventosa, N. Santo, R.    Rocca, L. Piergiovanni, J. Agric. Food Chem. 2012, 60, 782-790.-   ³ A. A. Krumnow, I. B. Sorokulova, E. Olsen, L. Globa, J. M.    Barbaree, V. J. Vodyanoy, J. Microbiol. Methods 2009, 78, 189-194.-   ⁴ S. Wu, J. Chen, Int. J. Biol. Macromol. 2013, 55, 254-257.-   ⁵ a) M. S. Hargrove, S. Krzywda, A. J. Wilkinson, Y. Dou, M.    Ikeda-Saito, J. S. Olson, Biochemistry 1994, 33,    11767-11775; b) M. R. Eftink, Biophysical Journal 1994, 66, 482-501.-   ⁶ S. Jahanshahi-Anbuhi, P. Chavan, C. Sicard, V. Leung, S. M. Z.    Hossain, R. Pelton, J. D. Brennan, C. D. M. Filipe, Lab Chip 2012,    12, 5079-5085; S. M. Z. Hossain, R. E. Luckham, A.-M. Smith, J. M.    Lebert, L. M. Davies, R. H. Pelton, C. D. M. Filipe, J. D. Brennan,    Anal. Chem. 2009, 81, 5474-5483-   ⁷ a) M. S. Hargrove, S. Krzywda, A. J. Wilkinson, Y. Dou, M.    Ikeda-Saito, J. S. Olson, Biochemistry 1994, 33,    11767-11775; b) M. R. Eftink, Biophysical Journal 1994, 66, 482-501-   ⁸ a) D. C. Carter, J. X. Ho, Adv Protein Chem 1994, 45,    153-203; b) G. Pico, Biochem Mol Biol Int 1995, 36, 1017-1023-   ⁹ S. Sitaula, S. D. Branch, M. F. Ali, Chemical Communications 2012,    48, 9284-9286; G. Jie, J. Yuan, J. Zhang, Biosensors and    Bioelectronics 2012, 31, 69-76; X. He, Z. Li, X. Jia, K. Wang, J.    Yin, Talanta 2013, 111, 105-110-   ¹⁰ a N. V. Padhye, M. P. Doyle, Applied and Environmental    Microbiology 1991, 57, 2693-2698; b R. D. Petty, L. A.    Sutherland, E. M. Hunter, I. A. Cree, Journal of Bioluminescence and    Chemiluminescence 1995, 10, 29-34

The invention claimed is:
 1. A method of performing a single step ormulti-step chemical reaction comprising: a) combining two or morereagents for the reaction, either separately or together, with anaqueous pullulan solution to provide reagent pullulan solutions or areagent pullulan solution, respectively; b) drying the reagent pullulansolutions or the reagent pullulan solution to provide solid polymericstructures or a solid polymeric structure, respectively; and c) if thetwo or more reagents are in separate solid polymeric structures in b),then treating the solid polymeric structures under conditions todissolve the solid polymeric structures and for the reagents to interactin a chemical reaction; or d) if the two or more reagents are togetherin the solid polymeric structure in b), then treating the solidpolymeric structure under conditions to dissolve the solid polymericstructure and for the reagents to interact in a chemical reaction,wherein the solid polymeric structures are or the solid polymericstructure is the components(s) of a sensor comprising two or morereagents for performing a chemical reaction entrapped in the same solidpolymeric structure or in different solid polymeric structures whereinthe solid polymeric structures are prepared from an aqueous solutioncomprising about 5% (w/v) to about 25% (w/v) of pullulan and at leastone of the two or more reagents is an enzyme.
 2. The method of claim 1,wherein the conditions to dissolve the solid polymeric structure(s)comprise contacting the solid polymeric structure(s) with water or anaqueous buffer.
 3. The method of claim 1, wherein, the two or morereagents are in separate solid polymeric structures and the solidpolymeric structures are stacked in layers on top of each other.
 4. Themethod of claim 3, wherein the layers are stacked in an order thatcorresponds to an order required to perform the chemical reaction. 5.The method of claim 1, wherein the two or more reagents are in separatesolid polymeric structures and the solid polymeric structures are in theshape of a pill, tablet or capsule, with each separate polymericstructure forming separate layers surrounding each other and the outerlayer of the pill, tablet or capsule comprises reagents that must reactfirst in the chemical reaction and the remaining layers are arrangedinternally to the outer layer in an order that corresponds to an orderrequired to perform the chemical reaction.
 6. The method of claim 3,wherein timing of dissolution of the layers is controlled to allowsufficient reaction time for each step of the chemical reaction.
 7. Themethod of claim 6, wherein the timing is controlled by one or more ofthickness of the layers and pullulan concentration.
 8. The method ofclaim 1, wherein the two or more reagents are in the same solidpolymeric structure.
 9. The method of claim 1, wherein the two or morereagents are in separate solid polymeric structures and the solidpolymeric structures are each dissolved in a single water or buffersolution to release the reagents for interaction in the chemicalreaction.
 10. A sensor comprising two or more reagents for performing achemical reaction entrapped in the same solid polymeric structure or indifferent solid polymeric structures wherein the solid polymericstructures are prepared from an aqueous solution comprising about 5%(w/v) to about 25% (w/v) of pullulan and at least one of the two or morereagents is an enzyme.
 11. The sensor of claim 10, wherein the two ormore reagents are entrapped in separate solid polymeric structures andthe solid polymeric structures are stacked in layers on top of eachother.
 12. The sensor of claim 10, wherein the two or more reagents areentrapped in separate solid polymeric structures and the solid polymericstructures are in the shape of a pill, tablet or capsule, with separatepolymeric structures forming separate layers surrounding each otherwherein a layer on the outside of the pill, tablet or capsule comprisesreagents that must react first in the chemical reaction and theremaining layers are arranged internally to the outside layer in anorder that corresponds to an order required to perform the chemicalreaction.
 13. The sensor of claim 10, wherein the two or more reagentsare entrapped in separate polymeric structures and the two or morereagents that are in separate polymeric structures comprise an enzymeand a substrate for the enzyme.
 14. The sensor of claim 10, wherein thetwo or more reagents are entrapped in the same solid polymericstructure.
 15. The sensor of claim 10, wherein one or more of the solidpolymeric structures are cast onto a substrate comprising at least oneof the two or more reagents for the chemical reaction.
 16. The sensor ofclaim 10, wherein the enzyme is DNA polymerases, restriction enzymes,DNA ligases, RNA ligases, luciferase, DNases, RNases, acetylcholineesterase, β-glucuronidase, β-galactosidase or lactate dehydrogenase. 17.The sensor of claim 10, wherein at least one of the two or more reagentsis an inorganic molecule selected from one or more of an inorganic acid,an inorganic base, an alkaline earth metal carbonate, an alkaline earthmetal sulfate, an alkali metal carbonate, an alkali metal sulfate and ametal complex.
 18. The sensor of claim 13, wherein the substrate for theenzyme is 5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-GLUC),5-bromo-4-chloro-3-indolyl-R-D-galactopyranoside (X-GAL),5-bromo-3-indolyl β-D-galactopyranoside (Bluo-Gla),5-bromo-6-chloro-3-indolyl β-D-galactopryaniside (Magenta-Gal),6-chloro-3-indolyl β-D-galactopyranoside (Salmon-Gal), 2-nitrophenylβ-D-galactopyranoside (ONPG) or 4-nitro β-D-galactopyranoside (PNPG).19. The sensor of claim 10, wherein the solid polymeric structures areprepared from an aqueous solution comprising about 10% (w/v) to about20% (w/v) of pullulan.
 20. The sensor of claim 10, wherein the two ormore reagents that are entrapped in different solid polymeric structurescomprise acetylcholine esterase (AChE) and indoxyl acetate.
 21. Thesensor of claim 10, wherein the two or more reagents in the same solidpolymeric structure comprise reagents for detection of adenosinetriphosphate (ATP).
 22. The sensor of claim 10, wherein the amount ofthe two or more reagents is premeasured in an amount needed to performthe chemical reaction.
 23. The sensor of claim 10, wherein the chemicalreaction produces a product that is detectable.
 24. The sensor of claim23, wherein the product is detectable using colorimetry, opticalspectrometry and/or fluorescence.
 25. The sensor of claim 24, whereinthe two or more reagents in the same solid polymeric structure comprisereagents for detection of adenosine triphosphate (ATP).
 26. The sensorof claim 25, wherein the reagents for ATP detection comprise luciferinand luciferase.
 27. The sensor of claim 25, wherein the reagents for ATPdetection comprise luciferin, luciferase, coenzyme A (CoA),dithiothreitol (DTT), a chelating reagent, MgCO₃ and MgSO₄.